Dissertations / Theses: 'Selenocystein'

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Mihara, Hisaaki. "Enzymological Studies of Cysteine Desulfurase and Selenocysteine Lyase." Kyoto University, 1999. http://hdl.handle.net/2433/78098.

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Kyoto University (京都大学) 0048 新制・課程博士 博士(農学) 甲第8031号 農博第1081号 新制||農||789(附属図書館) 学位論文||H11||N3326(農学部図書室) UT51-99-T742 京都大学大学院農学研究科農芸化学専攻 (主査)教授江﨑信芳, 教授清水昌, 教授關谷次郎 学位規則第4条第1項該当

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Tamura, Takashi. "Synthesis and Biochemical Function of Selenocysteine-containing Peptides." Kyoto University, 1993. http://hdl.handle.net/2433/168917.

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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである Kyoto University (京都大学) 0048 新制・課程博士 博士(農学) 甲第5495号 農博第775号 新制||農||660(附属図書館) 学位論文||H5||N2607(農学部図書室) UT51-93-R23 京都大学大学院農学研究科農芸化学専攻 (主査)教授左右田健次, 教授浅田浩二, 教授清水昌 学位規則第4条第1項該当

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Kossinova, Olga. "Insights into the selenocysteine incorporation mechanism in mammals." Strasbourg, 2011. http://www.theses.fr/2011STRA6221.

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L'acide aminé sélénocystéine est codé par UGA qui agit généralement comme un codon stop. Une machinerie spécialisée est utilisée pour incorporer cet acide aminé dans les sélénoprotéines qui implique une structure en tige-boucle, appelée SECIS, et des protéines. L'une d’elles est SBP2, SECIS Binding Protein 2. Pour mieux comprendre ce mécanisme et identifier de nouveaux partenaires de l'élément SECIS, deux stratégies ont été développées. 1/ identification des contacts de l’élément SECIS avec le ribosome par des pontages covalents. Dans ce but, j’ai construit des ARN messagers modèles, l’élément SECIS contenant des agents pontants. Après action des UV, il s’avère que SBP2 est liée au SECIS dans les complexes 48S et 80S de pré-translocation. Lorsque la formation de la liaison peptidique est bloquée par l’anisomycine, l’élément SECIS n’est plus lié à SBP2 mais à des protéines ribosomiques, SBP2 étant présente dans le complexe. L'interprétation est la suivante. Pendant la transpeptidation, SBP2 est associée au ribosome mais ensuite SBP2 le quitte et se lie au SECIS à l'étape de pré-translocation. 2/ Le site de liaison de SBP2 sur la sous-unité 60S n’avait pas encore été localisé. Les complexes SBP2-40S, 60S-SBP2 et 80S-SBP2 ont été soumis aux diépoxybutane ou 2-iminothiolane. Nous avons montré que SBP2 se lie à la sous-unité 60S uniquement et que l'ARNr 28S contribue davantage au site de liaison que les protéines ribosomiques. Pour identifier cette région de l’ARNr 28S, les radicaux hydroxyles ont été employés et nous avons montré que SBP2 réside sur le côté solvant de la sous-unité 60S, à proximité du site A The amino acid selenocysteine is encoded by a UGA triplet which acts generally as a stop codon. A specialized machinery is used to incorporate this amino acid into selenoproteins, involving a specific stem-loop, termed SelenoCysteine Insertion Sequence (SECIS), and some protein factors. One of those is the SECIS Binding Protein 2 (SBP2), which is necessary for ribosome recognition of the UGA as the Sec codon. Using synthetic selenocysteine mRNAs and translational inhibitors, several steps of mRNA translation were analyzed. The data obtained allowed us to propose the following mechanism for selenocysteine insertion : during the transpeptidation step of elongation, SBP2 is bound to the ribosome; however, after transpeptidation, SBP2 leaves the ribosome and binds the SECIS in the pre-translocation step. We showed earlier that SBP2 binds specifically to the purified human 60S but not to the 40S ribosome subunits but the actual location was unknown. The SBP2•40S, SBP2•60S and SBP2•80S complexes were thus studied using crosslinking reagents. SBP2 did not crosslink to the 40S subunit in either the 40S•SBP2 or 80S•SBP2 complexes, correlating with the binding data. However, SBP2 crosslinks to the 60S subunit in either the free state or in the 80S ribosome. I next showed that the 28S rRNA contributes more to the crosslink than ribosomal proteins. This led us to use hydroxyl radical footprinting to study the molecular environment of SBP2 on the ribosome. According to the probing data, the binding of SBP2 to the human 60S subunit protects 2 helices in expansion segment 7 of the 28S rRNA. I proposed that the SBP2 binding site is located in the vicinity of the L7/L12 stalk

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PIERRAT, STURCHLER CHRISTINE. "Etude structurale et fonctionnelle du trna selenocysteine eucaryote." Université Louis Pasteur (Strasbourg) (1971-2008), 1994. http://www.theses.fr/1994STR13209.

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L'aminoacide selenocysteine represente la forme biologique du selenium incorpore cotraductionnellement dans les selenoenzymes. Chez les procaryotes, l'incorporation de selenocysteine fait appel au trna selenocysteine, au codon uga situe dans un contexte particulier et a un facteur d'elongation specifique nomme selb. Le systeme eucaryote est moins bien connu et le but de ma these etait d'aider a une meilleure comprehension des mecanismes presidant au decodage de la selenocysteine chez les eucaryotes. Mes travaux de these ont porte essentiellement sur l'etude du trna selenocysteine eucaryote. J'ai, dans un premier temps, determine la structure secondaire et tertiaire de ce trna. Ceci a ete realise sur des trna transcrits in vitro, en utilisant une large panoplie de sondes de structure, enzymatiques et chimiques. La deuxieme partie de mon travail de these a consiste a etablir la liste des nucleotides modifies du trna#s#e#c en utilisant le systeme de microinjection de genes du trna#s#e#c ou de transcrits radioactifs dans les oocytes de xenopus laevis. J'ai montre notamment que u34, premiere base de l'anticodon du trna#s#e#c, est modifie en mcm#5u34, dans le cytoplasme des oocytes et ce, de maniere assez complexe. La derniere partie de mon travail de these porte davantage sur l'aspect fonctionnel de la molecule. Nous avons montre que les deux paires de bases non canoniques de l'helice acceptrice ainsi que la longueur exceptionnelle de l'helice d semblent avoir ete conservees au cours de l'evolution, pour la reconnaissance du trna par les facteurs agissant dans les etapes posterieures a la charge. Enfin, en collaboration avec l'equipe du dr. Bock, nous avons demontre que le trna#s#e#c eucaryote fonctionne de facon parfaitement correcte dans le systeme d'incorporation de selenocysteine chez les procaryotes

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Rogers, Sarah Elizabeth. "A selenocysteine containing αHL for single molecule studies". Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572843.

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Proteins containing selenocysteine (selenoproteins) have been found to exist in organisms from all domains of life. Selenoproteins are important for many in vivo processes such as the removal of reactive oxygen containing species (ROS), redox disulfide shuffling reactions, and pro-hormone activation. Structurally and functionally analogous to cysteine, selenocysteine's lower pKa appears to be the defining chemical difference between these two amino acids. Using a single-molecule electrical recording technique, rate constants for the reaction of selenocysteine with small molecule disulfides were obtained over a pH range of 6 - 10. Analogous single molecule ~riments carried out ~ .. - using cysteine, revealed that, after correcting for the ratio of selenolate to selenol and thiolate to thiol based on the pKa of each amino acid, the nuc1eophilicity of selenocysteine was comparable to that of cysteine. The selenium atom of the selenylsulfide bond was found to be substantially more electrophilic than a sui fur atom of the analogous disulfide bond and the leaving group ability of the selenolate of selenocysteine compared to the thiolate of cysteine were found to be comparable. Another biologically relavant interaction that occurs in vivo is the reaction between selenocysteine and organoarsenic (Ill) molecules. It is known that arsenic (Ill) compounds are toxic to organisms, and that this toxicity stems from the ability to coordinate to the thiol and selenol groups of the cysteine and selenocysteine residues within proteins. The reaction of selenocysteine with an organoarsenic species was investigated at the single molecule level over the pH range 6.5 - 8.5. By carrying out an analogous reaction between cysteine and the organoarsenic (Ill) species, it was found that selenocysteine and cysteine exhibit similar reaction rates. The organoarsenic reagent could exist in a range of different protonation states in solution and it was concluded that the rate of reaction was governed by the equilibrium of the arsenic molecule, where only some of the forms were reactive towards the selenocysteine and cysteine groups.

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Cassago, Alexandre. "Determinação estrutural da proteína Selenocisteína Sintase de Escherichia coli." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-01092010-114422/.

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A biossíntese do 21o. aminoácido, Selenocisteína (Sec - U), envolve uma complexa maquinaria enzimática composta, em eubactérias, pela Selenocisteína Sintase (SELA), Fator de Elongação de Selenocisteína (SELB), Selenofosfato Sintetase (SELD) e tRNA de Inserção Selenocisteína (tRNAsec). Em arqueobactérias e eucariotos existem ainda O fosforil tRNAsec Kinase (PSTK), SepSecS como SELA, EFSec como SELB, SPS1 e 2 como SELD e Proteína Ligante ao SECIS 2 (SBP2). O resíduo Selenocisteína é incorporado à proteína nascente no códon semelhante ao UGA de terminação identificado como local para incorporação de Sec, pela presença da Sequência de Inserção de Selenocisteína (SECIS), juntamente ao códon UGA na região codificante em bactérias e na região 3\'não codificante em arqueobactérias e eucariotos. SELA desempenha um papel central nessa via de biossíntese pela modificação do resíduo de Serina carregado ao tRNAsec pela enzima Seril-tRNA Sintetase (SerRS) convertendo-o em Selenocisteína. Essa enzima forma um complexo homodecamérico que reconhece e liga-se especificamente a SeriltRNAsec. A interação específica entre SELA e o tRNA permanece ainda não determinada. Nosso objetivo é a investigação estrutural por Espalhamento de Raios-X a Baixos Ângulos (SAXS) e cristalização da SELA e SELA-tRNAsec de Escherichia coli. Dados de SAXS determinaram parâmetros dimensionais como dimensão máxima, massa molecular e raio de giro. O modelo ab-inition foi calculado assumindo a simetria P52 de projeções de Microscopia Eletrônica de Transmissão (TEM). Os cristais obtidos do complexo SELA-tRNA mostraram o grupo espacial e dimensões da cela, apesar da baixa resolução dos dados. Para melhorar os estudos estruturais um modelo para proteína SELA de Escherichia coli foi construído usando o alinhamento da sequência de aminoácidos e o PDB, da proteína SELA putativa, de Methanococcus jannaschii, que apesar da baixa identidade resultou em um modelo muito bom. Adicionalmente, uma Análise de Acoplamento Estatístico (SCA) foi realizada baseada em alinhamentos múltiplos da proteína SELA, ordenando os aminoácidos mais conservados e a relação existente entre eles. The biosynthesis of the 21th amino acid, Selenocysteine (Sec - U), requires complex enzymatic machinery composed in eubacteria of: Selenocysteine Synthase (SELA), Selenocysteine Specific Elongation Factor (SELB), Selenophosphate Synthetase (SELD) and a specific Selenocysteine Inserting tRNA (tRNAsec). In archaeabacteria and eukaryotes there are O phosphoryl tRNAsec Kinase (PSTK), SepSecS as SELA, EFSec as SELB, SPS1 and 2 as SELD and SECIS Binding Protein 2 (SBP2). The Selenocysteine residue is incorporated into a nascent protein at a UGA like stop codon signaling as a Sec incorporation site by the presence of a Selenocysteine Insertion Sequence (SECIS), embedding the UGA codon in the coding region in bacteria and in a 3\' UTR in archaea and eukarya. SELA plays a central role in this pathway by modifying the Serine residue charged into the tRNAsec by Seryl-tRNA Synthetase (SerRS) and converting it into Selenocysteine. This enzyme forms a homodecameric complex that specifically recognizes and binds to Seryl-tRNAsec. The specific interaction of SELA and its tRNA remains unclear. Our aim is the structural investigation by Small Angle X ray Scattering (SAXS) and crystallization of Escherichia coli SELA and SELA-tRNAsec. SAXS datas determined dimensional parameters as maximum dimension, molecular mass and radius of gyration. Abinition model calculation was made assuming a P52 symmetry from Transmission Electron Microscope (TEM) projections Crystals of SELA-tRNA complex shown the space-group and cell dimensions, although its low resolution. To improve the structural studies a SELA model of E. coli was built using the amino acid sequences alignment and the PDB from Methanococcus jannaschii, SELA putative protein, which although the lower identities result in a very good model. In addition, a Statistical Coupling Analysis (SCA) was performed based on a multiple sequence alignment of SELA, ordering the most preserved amino acid and the relation between them.

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Manzine, Livia Regina. "Identificação de elementos estruturais no tRNAsecuca determinantes da ligação com proteínas." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-21032012-093929/.

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Em Escherichia coli a formação e incorporação do aminoácido selenocisteína é um evento cotraducional dirigido pelo códon de terminação UGA e deve se a uma complexa via de biosíntese cujas principais proteínas envolvidas são: Selenocisteína sintase (SELA), Fator de elongação de selenocisteína (SELB), Selenofosfato sintetase (SELD), Seril-tRNAser sintetase, um tRNA de inserção de selenocisteína (tRNAsec ou SELC) e uma sequência específica no RNA mensageiro, denominada de Sequência de inserção de selenocisteína (SECIS). A incorporação de selenocisteína em proteínas bacterianas inicia-se com a aminoacilação do tRNAsec com serina pela enzima Seril-tRNA sintetase formando seril-tRNAsec que é posteriormente convertido a selenocisteil-tRNAsec pela enzima SELA através de selenofosfato. Dessa forma, o trabalho teve seu foco estabelecido na realização de estudos bioquímicos e biofísicos da proteína SELA e na análise da interação dessa proteína com o ligante SELC para determinação de parâmetros de ligação envolvidos na formação desse complexo. O gene codificante para a proteína SELA foi subclonado, expresso em linhagem bacteriana WL81460(DE3) e a proteína SELA foi purificada como descrito na literatura; entretanto, uma nova metodologia para sua purificação foi desenvolvida proporcionando maior rapidez e rendimento. Estudos de filtração em gel, eletroforese nativa, focalização isoelétrica, dicroísmo circular, espectroscopia de fluorescência intrínseca e crosslinking químico proporcionaram uma melhor caracterização da proteína SELA e consequentemente uma maior compreensão de seu comportamento em solução. Ensaios de espectroscopia de anisotropia de fluorescência revelaram que a proteína SELA é capaz de se associar em estruturas superiores ao estado decamérico; essa análise pôde ser corroborada principalmente por dados de microscopia eletrônica empregando a técnica de negative staining. A metodologia de anisotropia de fluorescência também permitiu analisar a interação da macromolécula SELA com o ligante específico SELC, bem como com outros tRNAs mutantes possibilitando a realização de um mapeamento das regiões de SELC importantes para a interação. Além disso, essa técnica também foi satisfatoriamente empregada na determinação da estequiometria de ligação do complexo SELA-SELC revelando a proporção de 1 molécula de SELA para 10 tRNAs, o que contraria dados literários publicados em 1991 e 1992. The formation and incorporation of the amino acid selenocysteine in Escherichia coli is an event directed by cotraducional UGA codon and involves a complex biosynthesis pathway whose main proteins are: Selenocysteine synthase (SELA), elongation factor of selenocysteine (SELB), Selenophosphate synthetase (SELD), Seryl-tRNA synthetase, a selenocysteine tRNA (tRNAsec or SELC) and a specific sequence on the messenger RNA, called Selenocysteine insertion sequence (SECIS). The incorporation of selenocysteine in proteins of bacteria begins with the tRNAsec aminoacylation with serine by the enzyme Seryl-tRNA synthetase resulting in seryl-tRNAsec which is subsequently converted to selenocysteyl-tRNAsec by the enzyme Selenocysteine synthase (SELA). The selenium used in the conversion reaction is provided by Selenophosphate synthetase as selenophosphate and finally, the selenocysteyl-tRNAsec is delivered by the factor SELB to the ribosome. The present study focused on biochemical and biophysical studies of SELA protein and analysis of its interaction with the specific ligand (SELC) for determination of binding parameters involved in the formation of the complex. The gene coding for SELA protein was subcloned, expressed in WL81460(DE3) bacterial strain and the protein was purified as described in the literature; however a new, faster and more efficient method for its purification was developed. Studies of gel filtration, native gel electrophoresis, isoelectric focusing, circular dichroism, intrinsic fluorescence spectroscopy and chemical crosslinking provided a better characterization of SELA protein and a greater understanding of its behavior in solution. Analysis of fluorescence anisotropy spectroscopy revealed that SELA was able to associate in a supramolecular state. This analysis was mainly corroborated by data from electron microscopy employing negative staining technique. Fluorescence anisotropy methodology allowed us to analyse the interaction of SELA protein with the specific ligand SELC, as well as with others mutated tRNAs enabling a mapping of important regions in SELC for interaction. In addition, fluorescence anisotropy technique was also successfully used in determining the stoichiometry ratio of the complex SELA-SELC, showing a proportion of 1 molecule of SELA to 10 tRNAs, contraring to the literary data published in 1991 and 1992.

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Abe, Katsumasa. "Enzymatic studies of selenocysteyl-tRNA[Sec] biosynthesis." Kyoto University, 2008. http://hdl.handle.net/2433/136578.

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Kyoto University (京都大学) 0048 新制・課程博士 博士(農学) 甲第13489号 農博第1666号 新制||農||950(附属図書館) 学位論文||H20||N4314(農学部図書室) UT51-2007-T865 京都大学大学院農学研究科応用生命科学専攻 (主査)教授江﨑信芳, 教授植田和光, 教授渡邊隆司 学位規則第4条第1項該当

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Carlisle, Anne E. "Exploring the Role of Selenocysteine Biosynthesis Enzyme SEPHS2 in Cancer." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1112.

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Selenium is a micronutrient that is used by the selenocysteine biosynthesis pathway to produce the amino acid selenocysteine, which is required in selenoproteins. Many of the 25 human selenoproteins, such as glutathione peroxidases and thioredoxin reductases, play important roles in maintaining cellular redox homeostasis. In this study we characterize how this metabolic pathway is upregulated in cancer cells and how this increase in activity creates a unique vulnerability. We have outlined the evidence and underlying mechanisms for how many metabolites normally produced in cells are highly toxic, and we describe this concept as illustrated in selenocysteine metabolism. My thesis explores how SEPHS2, an enzyme in the selenocysteine biosynthesis pathway, is essential for survival of cancer, but not normal cells. SEPHS2 is required in cancer cells to detoxify selenide, an intermediate that is formed during selenocysteine biosynthesis. Breast and other cancer cells are selenophilic, owing to a secondary function of the cystine/glutamate antiporter SLC7A11 that promotes selenium uptake and selenocysteine biosynthesis, which, by allowing production of selenoproteins such as GPX4, protects cells against ferroptosis. However, this activity also becomes a liability for cancer cells because selenide is poisonous and must be processed by SEPHS2. These results show that SEPHS2 is a cancer specific target and indicates the therapeutic potential of SEPHS2 inhibition in the treatment of cancer. Collectively, this thesis identifies SEPHS2 as a targetable vulnerability of cancer cells, defines the role of selenium metabolism in cancer, and outlines a roadmap for future studies regarding toxic metabolites and cancer.

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Nelson, Samantha. "Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis." Master's thesis, University of Central Florida, 2014. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/6329.

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A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway. M.S. Masters Molecular Biology and Microbiology Medicine Biotechnology

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WALCZAK, ROBERT. "Decodage de la selenocysteine : relations structure-fonction dans l'element rna secis." Université Louis Pasteur (Strasbourg) (1971-2008), 1997. http://www.theses.fr/1997STR13150.

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Le seleniumest est present biologiquement sous la forme d'un acide amine particulier, la selenocysteine, qui est un analogue de la cysteine dans laquelle un atome de selenium remplace l'atome de soufre. La selenocysteine n'existe pas naturellement dans le pool des acides amines, mais est en fait synthetisee a partir de la serine par une voie tres complexe. La selenocysteine est ensuite incorporee cotraductionnellement dans les selenoproteines, en reponse au codon uga qui sert habituellement de signal de terminaison. Chez les eucaryotes, la presence d'un motif denomme secis, pour selenocysteine insertion sequence, localise dans la region 3' non traduite (3'utr) des mrna de selenoproteines, avait ete montree comme indispensable a l'incorporation cotraductionnelle de selenocysteine. La comparaison des sequences des mrna de plusieurs selenoproteines eucaryotes, corroboree par l'etude structurale avec des sondes enzymatiques et chimiques, nous a amene a proposer un modele de structure secondaire et tertiaire de l'element secis. Ce modele se caracterise par la presence de 2 helices (helice i et ii) separees par une bulle interne, l'helice ii etant surmontee par une boucle apicale. Le resultat le plus important reside en la mise en evidence d'un quartet de paires de bases non-watson-crick situees dans l'helice ii, a la sortie de la bulle interne. Ce quartet est compose en son centre d'un tandem de paires de bases g. A/a. G. L'etude structure-fonction du rna secis a ensuite valide notre modele de structure secondaire. Elle a aussi permis de confirmer l'arrangement structural du tandem central g. A/a. G predit par notre modele et d'etablir le role crucial joue par le quartet, notamment le tandem g. A/a. G, dans la translecture du codon uga. Nous avons egalement caracterise une proteine se liant a l'element secis du mrna de la glutathion peroxydase. Des experiences de competition de retard sur gel et de pontage aux uv ont montre que la fixation sur l'element secis de cette proteine de 60 a 65 kda, denommee sbp (pour secis binding protein), est specifique.

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Kwak, Mi-Sun. "Studies of protein systems depending on cysteine desulfurase and selenocysteine lyase." Kyoto University, 2005. http://hdl.handle.net/2433/144589.

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Kyoto University (京都大学) 0048 新制・課程博士 博士(農学) 甲第11835号 農博第1525号 新制||農||918(附属図書館) 学位論文||H17||N4084(農学部図書室) 23595 UT51-2005-K501 京都大学大学院農学研究科応用生命科学専攻 (主査)教授江﨑信芳, 教授清水昌, 教授坂田完三 学位規則第4条第1項該当

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HUBERT, NADIA. "Etude et identification de facteurs participant a l'insertion de selenocysteine chez les mammiferes." Université Louis Pasteur (Strasbourg) (1971-2008), 1998. http://www.theses.fr/1998STR13219.

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La selenocysteine represente la forme biologique majoritaire du selenium chez les eucaryotes. L'originalite de cet acide amine reside dans ses mecanismes de biosynthese et d'incorporation dans les proteines. En effet, la selenocysteine est synthetisee a partir de la serine par une voie specifique et tres complexe puis est insere au niveau d'un codon uga habituellement utilise comme signal de terminaison de la traduction. Chez les eucaryotes, les seuls elements connus du mecanisme d'incorporation de la selenocysteine sont le codon uga, en phase dans la region codante, le trna selenocysteine, et une structure en tige-boucle localisee dans la region 3 non traduite du rna messager, appelee secis (selenocysteine insertion sequence). Les proteines intervenant lors de cette reconnaissance n'ont pas encore ete toutes identifiees. L'identification de nouvelles proteines a donc constitue l'essentiel de mon sujet de these. Dans la premiere partie de ce memoire, nous nous sommes interesses a la fonction d'elements structuraux caracteristiques du trna#s#e#c dans les deux premieres etapes du mecanisme d'insertion de selenocysteine dans les proteines, la serylation du trna#s#e#c par la seryl-trna conventionnelle et la conversion du residu seryl-trna#s#e#c en selenocysteyl-trna#s#e#c par la selenocysteine synthase. Nous avons ainsi montre que la longueur exceptionnelle de la tige acceptrice de ce trna etait un determinant essentiel pour la reconnaissance du seryl-trna#s#e#c par la selenocysteine synthase. Dans le deuxieme volet de ce travail, nous avons entrepris l'identification de nouveaux facteurs impliques dans le mecanisme d'incorporation de selenocysteine. Nous avons ainsi pu mettre en evidence l'intervention de deux proteines differentes. La premiere a ete caracterisee par l'utilisation de sera autoimmuns provenant de patients atteints de polymyosite et reconnaissant une particule ribonucleoproteique contenant le trna#s#e#c. Cette particule contient une proteine de 55 kda, pouvant correspondre a un nouveau facteur de traduction, facteur d'elongation specifique du trna#s#e#c. La seconde proteine caracterisee par des experiences de retard sur gel et de pontage aux uv avec une sonde de rna secis, se fixe a l'element secis. Cette proteine, denommee sbp (secis binding protein) possede un poids moleculaire de 50 kda.

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Lacey, Brian. "Investigation Into the Role of the C-Terminal Vicinal Cysteine Residues in High MR Thioredoxin Reductases." ScholarWorks @ UVM, 2008. http://scholarworks.uvm.edu/graddis/130.

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Mammalian thioredoxin reductase (TR) contains the rare amino acid selenocysteine (Sec), which is essential for the enzyme’s catalytic activity. Substitution of the catalytic Sec residue for a cysteine (Cys) residue, results in a drop in kcat of 100- fold. Homologous high molecular weight TRs from other eukaryotes such as D. melanogaster and C. elegans, have naturally evolved a Sec to Cys substitution in their active sites and these enzymes function with high catalytic activity without the need for a Sec residue. Thus, various TRs can catalyze an identical reaction with either a Cys or Sec residue. A natural assumption in the field has always been that the lower nucleophilicity of a Cys thiol, relative to the selenol of Sec, is the reason for the much lower activity of the mammalian Cys-containing mutant. However, here I provide an alternative explanation. High Mr TRs contain either a Cys-Cys or Cys-Sec dyad that forms an eight-membered ring in the oxidized state during the redox cycle of the enzyme. These eight-membered ring structures are rare in protein structures, presumably due to the strain induced in the intervening peptide bond between the Cys residues. Here I take a “chemical approach” to studying the enzyme mechanism of TR by breaking it into two pieces. This approach is possible because of TR’s structural and mechanistic similarity to glutathione reductase (GR). In comparison to GR, TR contains an additional thiol-disulfide exchange step resulting from the presence of a sixteen amino acid C-terminal extension containing either a vicinal disulfide bond or vicinal selenylsulfide bond. This additional thiol-disulfide exchange step is in the form of the reduction and opening of the eight-membered ring motif. I have constructed a truncated version of the enzyme lacking the amino acid sequence possessing the ring motif so that I could isolate this ring-opening step from the rest of the catalytic cycle by using peptide disulfides/selenylsulfides as substrates. The results of this study using peptide substrates show that the ring opening step is the step of the catalytic cycle that is most effected by Sec to Cys substitution because the higher pKa of the Cys thiolate in comparison to the Sec selenolate means that the Cys residue must be protonated in this step.

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FAGEGALTIER, DELPHINE. "Recherche de nouveaux facteurs impliques dans le mecanisme d'incorporation de selenocysteine chez les eucaryotes." Université Louis Pasteur (Strasbourg) (1971-2008), 2000. http://www.theses.fr/2000STR13097.

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La selenocysteine est un analogue de la cysteine dans lequel l'atome de soufre est remplace par un atome de selenium. Les proteines qui contiennent de la selenocysteine (sec) sont impliquees dans des mecanismes d'oxydoreduction. Le residu selenocysteine joue un role central dans la catalyse. La selenocysteine est incorporee de maniere co-traductionnelle face a un codon uga. Ce mecanisme particulier est bien connu chez les eubacteries. Le propos de mon doctorat etait d'identifier des facteurs impliques dans le mecanisme d'incorporation de selenocysteine chez les eucaryotes. Nous avons recemment identifie le facteur d'elongation de souris mselb specialise dans l'insertion de selenocysteine. Nous avons montre que mselb : 1) fixe le gtp et le gdp de facon similaire a son homologue procaryote ; 2) reconnait specifiquement le sec-trna s e c in vivo et in vitro ; 3) est necessaire a l'incorporation de selenocysteine in vivo. Nous avons identifie des genes homologues chez l'homme, la drosophile, et c. Elegans. De plus, mselb forme un complexe specifique avec l'element secis uniquement en presence d'extraits totaux de cellules hela, montrant qu'au moins un autre facteur est implique dans ce mecanisme. Nous avons egalement recherche le cdna d'une proteine de 60 kda, sbp, qui se lie specifiquement a une structure en tige-boucle (secis) localisee dans la region 3 non traduite des mrna des selenoproteines, et necessaire a l'incorporation de selenocysteine. L'utilisation du systeme du triple hybride chez s. Cerevisiae, a permis d'isoler dbpb. Cependant, ce candidat n'a pas repondu a tous les criteres requis pour etre considere comme la proteine specifique sbp. Enfin, l'analyse structurale de nouveaux elements secis a montre que la majorite de ces structures en tige-boucle presente des appariements supplementaires dans la boucle apicale, de sorte qu'on peut les classifier en deux formes qui, de facon remarquable, correspondent a la famille de selenoproteines a laquelle ils appartiennent.

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Rodrigues, Elisandra Márcia. "Estudos moleculares das enzimas envolvidas na biossíntese de selenocisteína em Trypanosoma brucei e Leishmania major." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-05092008-090707/.

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Umas das principais formas biológicas de incorporação do selênio é na forma de um aminoácido denominado selenocisteína (Sec, U), que é incorporado co-traducionalmente ao polipeptídio nascente em posições específicas do códon UGA, que normalmente é reconhecido como códon de parada. A incorporação de selenocisteína em E. coli já está completamente esclarecida, com a participação dos genes que codifica para selenocisteína sintase (SELA), seril-tRNA sintetase (SerRS), um tRNASec específico (SELC), selenofosfato sintetase (SELD) e um fator de elongação próprio (SELB). Entretanto em eucariotos não há homólogos para SELA e existem evidências de haver a necessidade de dois passos enzimáticos que substituem a atividade desempenhada por SELA, com uma fosforilação da serina seguida de uma selenilação através das enzimas Fosfo-Seril-tRNASec Kinase (PSTK) e Sep-tRNA:Sec-tRNA sintase (SepSecS), respectivamente. A via de biossíntese e incorporação de selenocisteína é muito estudada em alguns organismos, mas ainda pouco explorada em Kinetoplastida. Nesse sentido, realizaram-se estudos moleculares das enzimas envolvidas nessa via, mais especificamente em Trypanosoma brucei e Leishmania major. Foram identificados o elemento SECIS na região 3´ do mRNA que atua no reconhecimento do códon UGA interno e, em fase de leitura na inserção de selenocisteína em Leishmania major e Leishmania infantum; a incorporação de Se75 em proteínas de Leishmania; a ocorrência do tRNASec em Trypanosoma e Leishmania e, adicionalmente todos os genes necessários para a síntese de selenocisteína: SELB, SELD, PSTK e SECp43. Foram obtidos clones dos genes selB e selD em vetor de expressão pET28a(+) e as proteínas foram expressas em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante SELD foi purificada em cromatografia de afinidade e seu pI e massa molecular foram determinados usando as técnicas de sistema Phast de eletroforese e gel nativo. As proteínas SELB, SELD, SECp43 e Seril tRNA sintetase foram imunolocalizadas no citoplasma de células nativas de T. brucei. Uma nova metodologia \"PTP tagging\" foi utilizada para estudos de interação protéica com uso de proteínas alvos SECp43, SELB e PSTK na busca de novas proteínas ligantes na via de selenocisteínas em T. brucei. Futuras investigações moleculares e estruturais das enzimas envolvidas na via de selenocisteína em Kinetoplastida poderão trazer informações relevantes no entendimento da biossíntese desse aminoácido, assim como possibilitar o desenvolvimento de inibidores específicos visando o tratamento de doenças causadas pelos parasitas Trypanosoma brucei e Leishmania major. One of the main biological forms of the selenium incorporation is the amino acid form named selenocysteine (Sec, U), which is incorporated co-translationally at the emerging new polypeptide in the specific positions at the UGA codon, that is usually recognized as stop codon. The incorporation of the selenocysteine in E.coli is already solved with the involvement of the genes that codify to selenocysteine synthase (SELA), seryl tRNA synthetase (SerRS), a specific tRNASec (SELC), selenophosphate synthetase (SELD) and a selenocysteine-specific translation elongation factor (SELB). However, in eukarya there is no SELA homologue, but there are evidences about the requirement of the two enzymatic steps that replace the activity performed by SELA, the fosforilation of the serine followed by selenocysteylation through the phosphoseryl-tRNASec kinase (PSTK) and Sep-tRNA:Sec-tRNA synthase (SepSecS) enzymes, respectively. Currently, the selenocysteine synthesis and its incorporation is more studied in many organisms, but less explored in Kinetoplastid. Subsequently, the molecular studies were done with the enzymes involved in this pathway, especially in Trypanosoma brucei and Leishmania major. The SECIS element was identified in the region 3´ of the mRNA, that acts in the recognition of the UGA codon positioned within a gene\'s open reading frame on the insertion of the selenocysteine in Leishmania major and Leishmania infantum; the incorporation of 75Se into Leishmania proteins, the occurrence of selenocysteine-tRNASec in both Leishmania and Trypanosoma; in addition, the finding of all genes necessary for selenocysteine synthesis, such as: SELB, SELD, PSTK, and SECp43. Clones were obtained from the selB and selD genes in the pET28a(+) expression vector and the enzymes were expressed in Escherichia coli BL21 (DE3). The recombinant SELD protein was purified by affinity chromatography and its pI and molecular mass were determined using: isoeletrophocusing electrophoresis and native gel. The proteins SELB, SELD, SECp43, and sery-tRNA synhetase were immune located in the cytoplasm in T. brucei native cells. A new methodology \"PTP tagging\" was utilized for protein interaction studies by using target proteins SECp43, SELB and PSTK to search new tagged proteins in selenocysteine T. brucei synthesis. Future molecular and structural investigation of the enzymes involved in Kinetoplastida selenocysteine biosynthesis will provide relevant information for understanding of the synthesis of this amino acid as well as the development of the specific inhibitors, focusing the treatment of the disease caused by Trypanosoma brucei e Leishmania major parasites.

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Evangelista, Jaqueline Pesciutti. "Estudos moleculares das enzimas Fosfoseril-tRNA sintease de Trypanosoma brucei e Leishmania major e Seril-tRNA sintease de Trypanosoma brucei." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-24072009-112648/.

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O estudo do processo de tradução no metabolismo celular atrai o interesse de vários grupos, em particular, o estudo do 21o aminoácido, a selenocisteína. A incorporação da selecisteína foi descrita em Escherichia coli e recentemente em eucariotos. O primeiro passo desta via é iniciado pela Seril-tRNA Sintetase que aminoacila o Ser-tRNASec (SelC) com uma serina. Em E. coli, o segundo passo é realizado pela Sec-sintetase (SelA) que remove o grupo hidroxil da cadeia lateral da serina, formando um intermediário aminoacrilil. Este serve como aceptor de seleno-fosfato gerando a selenocisteína. Em eucariotos, o processo análogo é realizado pela PSTK e pela SepSecS, que fosforila e seleniza a serina respectivamente. Interessados nesta parte da via, iniciamos estudos moleculares das enzimas Fosfoseril-tRNA Kinase de Trypanosoma brucei e Leishmania major e Seril-tRNA Sintetase de Trypanosoma brucei. Para o gene da enzima Fosfoseril-tRNA Kinase de T. brucei não foi possível obter um clone sem mutação. Já o gene da enzima Fosfoseril-tRNA Kinase de L. major foi clonado em vetor pET28 e a enzima foi expressa em células de E. coli porém com baixo rendimento impedindo a continuidade dos experimentos planejados. Portanto passou-se a investigar a enzima envolvida no primeiro passo da via, no caso, a Seril-tRNA Sintetase de T. brucei. Esta já se encontrava clonada e expressando em E. coli na fração solúvel. A proteína recombinante foi purificada com precipitação com 60% de sulfato de amônio e resinas de hidrofobicidade e de afinidade por níquel. Experimentos de gel nativo, DLS e fluorescência de anisotropia revelaram que, após a purificação, a enzima permanece estável e livre de agregações, possuindo um raio hidrodinâmico de 4,32nm e massa molecular de 110kDa. Acima de 150nM de proteína, ela encontra-se inteiramente na forma dimérica. Estabelecidos estes parâmetros, informações sobre a ligação com o Ser-tRNASec poderão ser obtidos a partir da técnica de anisotropia de fluorescência visto que experimentos iniciais realizados com a SerRS adicionando-se o Ser-tRNASec mostraram-se promissores. The translation process study is central role in the cellular metabolism and attracts the interest of several groups, in particular, the study of the 21º amino acid, the selenocystein. The selenocystein incorporation pathway was described in Escherichia coli and recently in eukaryotes. The first step of this pathway is initiated by Seryl-tRNA Synthetase that aminoacilates the Ser-tRNASec (SELC) with serine. In E. coli, the second step is performed by the Sec-synthase (SELA) that removes hydroxyl group of the serine side chain, forming an aminoacrylil intermediary. This serves as an acceptor of seleno phosphate generating the selenocystein. In eukaryotes, the similar process is performed by PSTK and SepSecS, which phosphorylate serine and adds the selenium, respectively. Interested in this pathway, we performed initial molecular studies of the Phosphoseryl-tRNA synthetase of Trypanosoma brucei and Leishmania major and Seryl-tRNA synthetase of Trypanosoma brucei. The gene that encodes T. brucei Phosphoseryl-tRNA synthetase was obtained with several mutations. However, the gene encoding the T. brucei Phosphoseryl-tRNA synthetase was cloned into pET28 vector and the enzyme was expressed in E. coli cells, however at low amounts hampering the intended experiments. Therefore we initiated the investigation of the enzyme involved in the first step of this pathway, the Seryl-tRNA Synthetase from T. brucei. The enzyme was already cloned and expressing in the soluble fraction of E. coli. The recombinant protein was purified using 60% ammonium sulfate precipitation, hydrophobic and nickel affinity chromatography. Native gel experiments, DLS and anisotropy fluorescence was performed and allowed to conclude that, after purification, the enzyme remains stable and free of aggregation, with a hydrodynamic radius of 4.32 nm, molecular weight of 110kDa. Above 150nM protein its entirely in the dimeric form. Information about Ser-tRNASec binding can now be obtained from the technique of anisotropy seen that initial experiments with SerRS add Ser-tRNASec be shown to be promising.

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24

Barry, Amanda Nell. "Spectroscopic studies of the human copper chaperone for superoxide dismutase : probing the active cluster with selenocysteine variants." Full text open access at:, 2007. http://content.ohsu.edu/u?/etd,258.

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25

Ganichkin, Oleg [Verfasser], C. Wahl [Akademischer Betreuer] Markus, Ralf [Akademischer Betreuer] Ficner, Detlef [Akademischer Betreuer] Doenecke, and Claudia [Akademischer Betreuer] Höbartner. "Crystal structure analysis of selenocysteine biosynthesis components / Oleg Ganichkin. Gutachter: Ralf Ficner ; Detlef Doenecke ; Claudia Höbartner. Betreuer: C. Wahl Markus." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2010. http://d-nb.info/1042532729/34.

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Ganichkin, Oleg Verfasser], C. Wahl [Akademischer Betreuer] Markus, Ralf [Akademischer Betreuer] Ficner, Detlef [Akademischer Betreuer] [Doenecke, and Claudia [Akademischer Betreuer] Höbartner. "Crystal structure analysis of selenocysteine biosynthesis components / Oleg Ganichkin. Gutachter: Ralf Ficner ; Detlef Doenecke ; Claudia Höbartner. Betreuer: C. Wahl Markus." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2010. http://d-nb.info/1042532729/34.

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27

Santesmasses, Ruiz Didac 1978. "Selenoproteins across the tree of life: Methods and applications." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/565634.

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La selenocïsteina és coneguda com a l'aminoàcid 21. Les selenoproteïnes incorporen selenocïsteina en resposta a codons UGA específics mitjançant un mecanisme de recodificació, el qual és present en els tres dominis de la vida, però no en tots els organismes. Els programes estàndard per a la predicció de gens consideren UGA només com a codó stop, per aquesta raó l'anotació de selenoproteínes és, generalment, incorrecte. Hem desenvolupat mètodes computacionals per a la predicció de selenoproteïnes. Mitjançant l'aplicació d'aquestes i altres eines, hem caracteritzat selenoproteïnes a través de l'Arbre de la Vida, on hem observat una evolució dinàmica en la utilització de selenocïsteina en els diferents llinatges. Hem caracteritzat l'abundància i distribució de selenoproteïnes en el microbioma humà. Hem caracteritzat les selenoproteïnes presents a Lokiarchaeota, les quals presenten trets eucariòtics. Finalment hem dedicat especial atenció als insectes, en els quals una progressiva reducció en el nombre de selenoproteïnes culminà en múltiples extincions de selenoproteïnes en esdeveniments evolutius independents. Selenocysteine is known as the 21st amino acid. Selenoproteins incorporate selenocysteine in response to specific UGA codons through a recoding mechanism, which present in the three domains of life, but not in all organisms. Standard gene prediction programs consider UGA only as stop, and selenoproteins are normally misannotated. We have developed computational methods for prediction of selenoproteins. By applying these and other tools, we have characterized selenoproteins across the Tree of Life, showing a diverse evolution of the utilization of selenocysteine in different lineages. We have characterized the abundance and distribution of selenoproteins in the human microbiota. We characterized the selenoproteins in Lokiarchaeota, which have some eukaryotic-like features. Finally we gave special attention to insects, in which a progressive reduction in the number of selenoproteins culminated in multiple independent selenoprotein extinctions.

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Faim, Lívia Maria. "Estudos estruturais e funcionais da Selenofosfato Sintetase de Trypanosoma brucei e Leishmania major." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-31072014-165101/.

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A síntese e incorporação de Selenocisteína em selenoproteínas ocorre co tradicionalmente direcionado pelo códon de terminação UGA. Uma maquinaria única de enzimas e fatores proteicos é necessária para síntese de selenocisteína e decodificação do códon UGA de terminação da tradução para inserção de selenocisteína. Dentre as enzimas envolvidas, está a Selenonofosfato sintetase (SPS2), responsável por catalisar a ativação de seleneto com adenosina 5 trifosfato (ATP) para gerar selenofosfato, o doador de selênio reativo que é substrato da próxima enzima da via para formação de selenocisteína. Estudos recentes identificaram a presença da via de biossíntese de selenocisteína em parasitas kinetoplastidas e subsequentemente a proteína SPS2 de Trypanosoma brucei e Leishmania major foram caracterizadas. Entretanto, trabalhos estruturais e funcionais das enzimas permaneceram não reportados. Dessa forma, este trabalho teve seu foco estabelecido na realização de estudos estruturais e funcionais da SPS2 de T. brucei e L. major. Para caracterização da proteína em solução foram empregadas as técnicas de cromatografia de exclusão de tamanho, eletroforese em gel nativo, espalhamento dinâmico de luz (DLS), espalhamento de Raios X a baixo ângulo (SAXS) e ultracentrifugação analítica (AUC). Os resultados obtidos revelaram uma mistura de dímeros e tetrâmeros em solução para ambas SPS2 com predominância de dímeros. Muitas estratégias de cristalização e melhorias na difração foram utilizadas para obtenção de cristais proteicos apropriados para determinação da estrutura cristalográfica das SPS2. Cristais de SPS2 de T. brucei inteira e SPS2 de L. major com N-terminal truncado foram obtidos. Porém, somente a estrutura cristalográfica da proteína SPS2 de Leishmania major com o N-terminal truncado a 1,9 Å de resolução foi determinada. Estudos comparativos entre esta estrutura e outras selenofosfato sintetases mostrou a mesma organização estrutural entre elas. Experimento de complementação funcional das SPS2 truncadas e mutadas pontualmente revelou três resíduos localizados no N-terminal como fundamentais para atividade da SPS2 (Leu33, Thr34; Tyr36 e Leu37, Thr38; Tyr40 para SPS2 de T. brucei e L.major, respectivamente). Análise mutacional baseada nas estruturas cristalográficas indicou que estes resíduos podem estar envolvidos no mecanismo de entrega do selenofosfato para a próxima enzima da via, a Selenocisteína sintase. Isto poderia evitar a difusão de compostos reativos de selênio, resultando em uma eficiência na síntese de selenocisteína. Os resultados aqui apresentados forneceram informações importantes e novas perspectivas a respeito do mecanismo de catalise da enzima selenofosfato sintetase na via de síntese de selenocisteína. The synthesis and incorporation of selenocysteine in selenoproteins occurs cotranslationally directed by the UGA stop codon. An unique machine of enzymes and protein factors are required for selenocysteine synthesis and decoding of UGA translation termination codon for the insertion of selenocysteine. Among the enzymes involved, Selenonofosfato synthetase (SPS2) is the responsible for catalyzing the activation of selenite with adenosine 5\' - triphosphate (ATP) to generate selenophosphate, the reactive selenium donor, which is substrate of the next pathway enzyme to formation of selenocysteine. Recent studies have identified the presence of selenocysteine biosynthesis in parasites Kinetoplastidas and subsequently, the SPS2 protein of Trypanosoma brucei and Leishmania major have been characterized, however, structural and functional studies of enzymes remain not reported. Thus, this present work report biochemical and biophysical studies of SPS2. To characterize the protein in solution, there were employed the techniques of size exclusion chromatography, native gel electrophoresis, dynamic light scattering (DLS), Small angle X-ray scattering angle (SAXS) and analytical ultracentrifugation (AUC). The results revealed a mixture of dimmers and tetramers in solution for SPS2 with predominance of dimers. Many strategies and improvements in crystallization and diffraction were used to obtain suitable SPS2 crystals for determination of the crystallography structure. T. brucei SPS2 crystals and L. major SPS2 crystals with truncated N-terminal were obtained. However, only the structure of SPS2 protein from L. major with truncated N-terminal to 1.9 Å of resolution was solved. Comparative studies of this structure with other selenophosphate synthases revealed the same structural organization. Functional complementation experiments of truncated and mutated SPS2 revealed three residues located in the SPS2 N- terminal as essential for the activity of the enzyme (Leu33 , Thr34 and Tyr36 to T. brucei SPS2; Leu37 , Thr38 and Tyr40 to L. major SPS2) . Mutational analysis based on the crystal structures indicated that these residues may be involved in the mechanism of selenophosphate delivery to the pathway enzyme next, the selenocysteine synthase. This found could prevent the diffusion of reactive selenium, resulting in selenocysteine synthesis efficient. The results presented here provided important information and new insights about the of selenophosphate synthetase catalysis mechanism in the selenocysteine synthesis pathway.

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Bellini, Natalia Karla. "Estudo celular, bioquímico e biofísico da enzima selenofosfato sintetase de Naegleria gruberi." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-17092015-105838/.

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O microrganismo alvo deste estudo pertence ao gênero Naegleria, que compreende amebas de vida livre amplamente distribuídas ao redor do mundo. Estas possuem estratégias de adaptação em condições de temperatura e pH que envolvem a diferenciação das células para as formas flagelada e cística. A via de biossíntese e incorporação do aminoácido selenocisteína (Sec, U) em N. gruberi foi descrita e, devido à incorporação co-traducional deste aminoácido em resposta a um códon UGA em fase de leitura, possui diversos fatores específicos que tornam a via alvo de estudos moleculares. Dentre os genes identificados, destaca-se o de selenofosfato sintetase (SPS), uma proteína funcionalmente dimérica envolvida na catálise da conversão de seleneto e adenosina 5´-trifosfato (ATP) em selenofosfato, essencial à síntese de Sec. Diferindo das SPSs homólogas, em N. gruberi a proteína (NgSPS2) é codificada em fusão N-terminal com uma metiltransferase e totaliza 737 aminoácidos. Esta descoberta motivou os objetivos da pesquisa baseada na investigação celular de NgSPS2 nativa nas três diferentes formas de vida de N. gruberi através de ensaios imunoenzimáticos, e a caracterização bioquímica e biofísica da proteína recombinante. A análise dos resultados obtidos por Western blot indicaram que NgSPS2, in vivo, apresenta os dois domínios metiltransferase e SPS separados após a tradução para uma cultura amebóide e, após alcançar a diferenciação de cada uma das formas isoladamente, este resultado se confirmou também para cistos e flagelados. A investigação de N. gruberi em cultura indica o aumento na atividade da via de síntese de selenoproteínas na presença de selênio conferindo resistência às condições de estresse oxidativo. A caracterização bioquímica do domínio C-terminal de NgSPS2, por cromatografia de exclusão molecular analítica e eletroforese não desnaturante, revelou predominância de dímeros em solução, coerente com SPSs homólogas. Os testes de cristalização não resultaram na obtenção de cristais, porém a proteólise limitada permitiu selecionar tripsina como potencial para a clivagem do N terminal do N terminal flexível. A conservação dos resíduos de aminoácidos funcionais em NgSPS2.CTD e seu comportamento em solução confirmam a obrigatoriedade da união de cada monômero e, por isso o domínio metiltransferase adicional pode ser desfavorável à montagem do dímero e in vivo a fusão é desfeita após a tradução. The target microorganism of the present study belongs to the Naegleria genus. This genus includes free life amoebas widely distributed around the world that, in order to survive in bad temperature and pH environments, developed an adaptive strategy consisting of cells differentiation to flagellate and cystic form. The biosynthesis and incorporation of selenocysteine amino acid (Sec, U) in N. gruberi has been described and, because of the co-translational incorporation of this amino acid in response to a UGA codon during the reading step, this process has several specific factors which make it a target for molecular studies. Among the identified genes, we can highlight the one which encodes the selenophosphate synthetase that is involved in the catalytic conversion of selenite and adenosine triphosphate into selenium phosphate, a necessary step to the Sec synthesis that uses selenide and ATP to produce selenophosphate. SPS from N.gruberi is encoded with an methyltransferase N-terminal fused with the typical SPS C-terminal domain, an open read frame that contains 2211 nucleotides encoding 737 amino acids. This discovery has motivated the initial aims of this project, based on the cellular investigation of SPS2, native on the three different form lifes of N. gruberi, through immunoenzymatic assays, besides a study with the recombinant protein to clarify the biochemistry and biophysics features of NgSPS2. The results indicated that the protein do not keep both domains fused after the translation process, suggesting that they need to be separated to perform their biological function. The investigation of the N. gruberi culture revealed that the cells become less sensitive to stress agent in the presence of selenium, which seems to be correlated with the increasing activity of the selenoprotein synthesis. The biochemistry characterization of the NgSPS2 C-terminal domain, using size exclusion chromatography and electrophoresis under non-denaturing conditions revealed the predominance of dimers in solution according with the typical homologous SPS oligomeric state. The crystallization tests have not resulted in crystal growth; however, the limited proteolysis may be an alternative to optimize the crystallization process. These studies may enlarge the knowledge about the biosynthesis of Sec. in N. gruberi.

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Kotini, Suresh Babu [Verfasser], Marina [Akademischer Betreuer] Rodnina, Holger [Akademischer Betreuer] Stark, and Ralf [Akademischer Betreuer] Ficner. "Molecular mechanism of selenocysteine incorporation in bacterial translation / Suresh Babu Kotini. Gutachter: Marina Rodnina ; Holger Stark ; Ralf Ficner. Betreuer: Marina Rodnina." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2012. http://d-nb.info/104359468X/34.

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Barber, Drew. "Selenium In Thioredoxin Reductase: Resistance To Oxidative Inactivation, Oxidation States, And Reversibility Of Chemical Reactions." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/943.

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Selenium is a required trace element which was originally discovered by the Swedish chemist Jons Jacob Berzelius in 1817. It was initially believed to be a toxin as it was identified as being the cause of hoof maladies and excessive hair loss in horses that feed upon plants with high selenium content. It wasn’t until 1957 that the potential contributions of selenium to physiology were first demonstrated. Selenium is now known to play a critical role in the maintenance of human health. Interestingly, unlike other trace metals/semi-metals, selenium is directly incorporated into proteins in the form of the amino acid selenocysteine (Sec) in a very complicated and energetically costly fashion. Though rare, being found in only 25 human proteins, Sec proteins are involved in numerous vital biological processes including maintenance of redox homeostasis and anti-oxidant defense. Even though Sec is essential, the reason that Sec replaces its structural analog cysteine (Cys) in only 25 proteins is not widely agreed upon. A previous model suggests that the replacement of Cys with Sec provides enzymes with a type of catalytic advantage. The presence of Cys-containing orthologs of mammalian Sec-enzymes in other eukaryotes argues against this model. A newer model to explain the use of Sec is that the gain of function imparted to an enzyme by replacing Cys with Sec is the ability of Sec to impart chemical reversibility. Building on previous results from our lab demonstrating the ability of Sec to confer proteins with the ability to resist over oxidation we have elucidated the mechanism by which Sec containing thioredoxin reductase (TrxR) resists over oxidation. The ability of Sec-TrxR to resist oxidative inactivation is due to the greater electrophilicity of Sec relative to Cys. This allows for quicker resolution and prevents over oxidation. Based on these findings we also investigate the utility of the alkylating agent dimedone to probe the oxidation state of Sec. Interestingly, it was discovered that dimedone will react with seleneninic acid with the resulting adduct being labile. Additonally it was discovered that dimedone will also react with seleninic acid, resulting in the formation of a dimedone dimer. These results call into question the usefulness of dimedone in deteremining the oxidation state of Sec. Finally, we provide evidence that Sec-TrxR enzymes are able to catalyze single electron reductions. This is most likely due to the formation of a stable Sec radical intermediate. As a whole this project provides support for the theory that Sec was selected for due to its ability to convey chemical reversiablity to proteins.

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Serrão, Vitor Hugo Balasco. "Complexos macromoleculares da via específica de incorporação de selênio de Escherichia coli." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-20032013-091148/.

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A existência de uma maior variedade de aminoácidos codificados pelo código genético tem estimado estudos sobre os mecanismos de síntese, reconhecimento e incorporação desses resíduos nas cadeias polipeptídicas nascentes. Um exemplo é a via de incorporação de selenocisteína evento cotraducional dirigido pelo códon UGA. Em bactérias, essa via conta com uma complexa maquinaria molecular composta por: Selenocisteína Sintase (SelA), Fator de Elongação Específico de Reconhecimento (SelB), Selenofosfato Sintetase (SelD), tRNA específico (SelC ou tRNAsec), sequência específica no mRNA (Sequência de Inserção de Selenocisteínas - SECIS) e Aminoacil tRNA Sintetase (aaRS). Pelo fato do selênio ter uma toxicidade elevada em ambientes celulares, é fundamental a compreensão do mecanismo catalítico e razão estequiométrica na formação dos complexos da via na etapa de incorporação junto ao tRNAsec, bem como sua caracterização estrutural foram os objetivos deste trabalho. A proteína SelA foi expressa e purificada para utilização em análises envolvendo microscopia de força atômica, microscopia eletrônica de transmissão com contraste negativo e em gelo vítreo foram realizadas nos complexos SelA e SelA-tRNAsec, visando obter um modelo estrutural e a razão estequiométrica dos complexos. A fim de compreender o mecanismo de passagem do selênio, ensaios de anisotropia de fluorescência e de microcalorimetria, corroborados pelas análises de troca de hidrogênio-deutério acoplado a espectrometria de massa e espectroscopia de infravermelho, elucidaram a formação e estequiometria do complexo ternário SelAtRNA sec-SelD. Tentativas de cristalização e análises cristalográficas também foram realizadas, no entanto, sem sucesso. Com os resultados obtidos foi possível propor que o reconhecimento de SelD e, consequentemente, a entrega do selenofosfato, seja uma etapa crucial da via de incorporação de selenocisteínas. The existence of a greate variety of amino acids encoded by the genetic code has stimulated the study of the mechanisms of synthesis, recognition and incorporation of these residues in the nascent polypeptide chains. An example of genetic code expansion is the selenocysteine incorporation pathway an event cotraducional by the UGA codon. In bacteria, this pathway has a complex molecular machinery comprised: Selenocysteine Synthase (SelA), Specific Elongation Factor (SelB), Selenophosphate Synthetase (SelD), tRNA-specific (SelC or tRNAsec), Specific mRNA Sequence (SElenocysteine Insertion Sequence - SECIS) and Aminoacyl tRNA Synthetase (aaRS). Because selenium has high toxicity in cellular environments; it is essential for cell survival the association of this compound with proteins, in this case, selenoprotens and the associated proteins involved in the selenocysteine synthesis. Therfore the understanding of the catalytic mechanism, stoichiometric ratio, protein complex formation with the tRNAsec, and its structural characterization were the objectives of this work. The SelA protein was expressed and purified to used in analyzes involving atomic force microscopy, transmission electron microscopy with negative stain and in vitreous ice were performed in the complex SelA and SelA-tRNAsec in order to obtain a structural model of the complex and the stoichiometric ratio of its components. To study the selenium association with protein of the synthesis pathway, fluorescence anisotropy assays and isothermal titration calorimetry corroborated by the analysis hydrogen-deuterium exchange coupled to mass spectrometry and infrared spectroscopy were employed.Crystallization attempts were made and preliminary crystallographic analyzes were also performed, however, so far unsuccessfuly. The results obtained were possible to develop the hypothesis about the SelD recognition and, consenquently, the selenophosphate delivery, a crucial stage of the selenocysteine incorporation pathway.

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Silva, Ivan Rosa e. "Estudos biofísicos da Selenofosfato Sintetase de Escherichia coli e investigação de seu papel na via de biossíntese de Selenocisteínas." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-22032012-084829/.

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A principal forma biológica do selênio em vários organismos é o aminoácido Selenocisteína (Sec, U), que é incorporado em um polipeptídio emergente em códons UGA específicos. Em Escherichia coli, esta incorporação requer os genes que codificam para Seril-tRNA Sintetase (SerRS), Selenocisteína Sintase (SELA), um tRNASec específico (SELC), Selenofosfato Sintetase (SELD) e um fator de elongação de transcrição específico (SELB). A proteína Selenofosfato Sintetase (EC 2.7.9.3) pertence à família AIRS, de proteínas que têm o ATP como substrato, e produz o composto biologicamente ativo doador de selênio, o monoselenofosfato, a partir de ATP e seleneto. O gene selD em E. coli tem 1041 pares de bases e codifica uma proteína com 347 aminoácidos e massa molecular de 37 kDa. A fase aberta de leitura do gene selD foi amplificada do DNA genômico de E. coli e clonada em vetor de expressão pet28a(+) (Novagen). A proteína recombinante foi superexpressa em E. coli por indução com IPTG e purificada por cromatografia de afinidade por ligação a metal e a fração eluída foi concentrada por ultrafiltração. Em seguida, o produto foi submetido à clivagem da cauda de histidinas com Trombina. Para purificar o produto de reação de clivagem com protease e para estimar sua massa molecular e estado oligomérico, empregou-se cromatografia de exclusão molecular. A proteína pura foi utilizada em experimentos de Gel Nativo e em estudos das suas propriedades hidrodinâmicas realizados por meio de Espalhamento Dinâmico de Luz (DLS), Espalhamento de Raios-X a Baixo Ângulo (SAXS) e Ultracentrifugação Analítica (AUC). Os resultados obtidos revelam uma mistura de oligômeros em solução, em um equilíbrio dímero-tetrâmero e tetrâmero-octâmero. Um modelo tridimensional para o homodímero de SELD de E. coli foi obtido por Modelagem Molecular e suas propriedades hidrodinâmicas preditas concordam com aquelas obtidas experimentalmente. Adicionalmente, triagens de condições de cristalização da proteína revelaram condições em que a proteína cristaliza na forma de pequenas agulhas e ensaios de otimização por variação da concentração de agente precipitante e pH não resultaram em monocristais adequados para difração de raios-X. A análise do papel da SELD na via de biossíntese de Selenocisteínas levanta a hipótese de que esta proteína deve entregar o monoselenofosfato para o complexo SELA-SELC de modo que o selênio seja incorporado para formação do aminoácido Selenocisteína, já que os compostos de selênio são tóxicos quando estão livres na célula. Portanto, a investigação da interação da SELD com o complexo SELA-SELC foi observada pelo monitoramento da anisotropia de fluorescência do complexo SELA-SELC mediante titulação de SELD. A análise local da interação para manutenção do complexo SELD-SELA-SEC foi feita por meio de espectrometria de massas com troca H/D, que revelou possíveis sítios de interação na superfície da SELD. Os resultados mostrados neste trabalho ampliam o conhecimento sobre a via de biossíntese de Selenocisteína, revelando detalhes da interação da SELD com o complexo SELA-SELC. The main biological form of selenium in several organisms is the amino acid Selenocysteine (Sec, U), which is incorporated into selenoproteins in specific UGA codons. In Escherichia coli, it requires the genes that codify to Seryl-tRNA Synthetase (SerRS), Selenocysteine Synthase (SELA), a specific tRNASec (SELC), Selenophosphate Synthetase (SELD) and a specific translation elongation factor (SELB). Selenophosphate Synthetase (EC 2.7.9.3) belongs to AIRS superfamily of proteins that have ATP as a substrate and this protein produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide. The selD gene from E. coli is 1041 base pairs long and codifies a protein with 347 amino acids and molecular mass of 37 kDa. The open reading frame of selD gene was amplified from E. coli genomic DNA and cloned into pET28a(+) expression vector (Novagen). The recombinant protein was overexpressed in E. coli by IPTG induction and purified by metal affinity chromatography, and the eluted fraction was concentrated by ultrafiltration. The product was used for Thrombin protease cleavage of the 6-His tag. In order to purify the product of proteolysis and to estimate its molecular mass and oligomeric state, we used size exclusion chromatography. The pure protein sample was used for Native Gel Electrophoresis. Hydrodynamic properties of the protein were studied by Dynamic Light Scattering (DLS), Small angle X-ray scattering (SAXS) and Analytical Ultracentrifugation (AUC). The results show an equilibrium between SELD oligomeric forms, as dimer-tetramer and tetramer-octamer association in solution. A tridimensional model of E. coli SELD was obtained by Molecular Modelling and its predicted hydrodynamic properties agree with those observed experimentally. In addition, crystal screening revealed crystallization conditions suitable for protein crystallization as small needles, but optimization of these conditions by precipitant agent and pH variation did not result in monocrystals reliable for X-ray diffraction. An analysis of SELD´s role in the Selenocysteine biosynthesis pathway indicates that SELD must deliver monoselenophosphate to the SELA-SELC complex so that the selenium is incorporated to the amino acid to form selenocysteyl-SEC, since selenium compounds are toxic when they are freely available in the cell. This interaction was observed by fluorescence anisotropy. The local analysis of complex formation was monitored by mass spectrometry after H/D exchange and revealed possible sites for this interaction on SELD surface. The results improve our knowledge about the Selenocysteine pathway in the cell, showing details of the interaction between SELD and the SELA-SELC complex.

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Martil, Daiana Evelin. "Estudos estruturais da Seril-tRNA Sintetase nativa e em interação com tRNAs cognatos de Trypanosoma brucei." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-31072014-161157/.

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A síntese de selenocisteína e sua incorporação co-traducional em selenoproteínas como resposta a um códon UGA em fase requerem uma complexa maquinaria molecular. Em eucariotos, foram identificados componentes que participam da reação de formação de selenocisteína: Seril-tRNA sintetase (SerRS), O-fosfoseril-tRNA quinase (PSTK), SECIS Binding Protein 2 SBP2, um fator de elongação específico para Sec (EFSec), selenofosfato sintetase 1 (SPS1) e selenofosfato sintetase 2 (SPS2), SEPSECS, proteína ligante de RNA SECp43, proteína ribossomal L30, um tRNA de inserção de selenocisteína (tRNASec, SELC) e uma sequência específica no RNA mensageiro (elemento SECIS). O primeiro passo da incorporação de selenocisteína em proteínas é realizado pela SerRS, que aminoacila o tRNA com serina através da ativação da serina por Mg+2 e ATP, levando a formação de um intermediário ligado a enzima (Ser-AMP). Posteriormente, ocorre a mudança do radical Ser do intermediário Ser-AMP para o tRNASec, e subsequentemente, a conversão enzimática de Ser-tRNASec para Sec-tRNASec. Através de análises in sílico nosso grupo identificou componentes da maquinaria de inserção de selenocisteína em espécies de Kinetoplastida. Foram identificados homólogos de tRNASec e as enzimas TbSerRS, TbSPS2, TbPSTK, TbSepSecS e TbEFSec. Nosso principal alvo é o estudo estrutural da SerRS de Trypanosoma brucei nativa e em complexo com o tRNASec e com as isoformas do tRNASer. Uma nova metodologia no processo de purificação desta enzima foi desenvolvida e, através das técnicas de cromatografia de exclusão molecular, espalhamento de luz dinâmico e ultracentrifugação analítica conseguimos determinar o estado oligomérico da TbSerRS. O resultado de dímeros em solução corroborou com dados reportados na literatura, além de verificarmos por meio de estudos de cinética enzimática que a enzima encontra-se ativa sob as condições utilizadas. A técnica de ultracentrifugação analítica de sedimentação em equilíbrio também nos permitiu verificar a formação do complexo SerRS-tRNA, mas não nos possibilitou definir a estequiometria deste complexo. Estudos estruturais da enzima nativa e em interação com os tRNAs SELC e com as isoformas do tRNASer, L-serina, um análogo não hidrolisável de AMP, MgCl2, e com porções menores dos tRNAs foram realizados por meio da cristalografia por difração de raios X. Através dessa técnica, dezessete conjunto de dados foram coletados, processados e estão em fase de refinamento. Algumas análises estruturais possibilitaram confirmar a presença de duas moléculas de glicerol em cada monômero na região do sítio ativo para a estrutura da TbSerRS nativa e uma molécula de dAMP para o complexo TbSerRS-dAMP. The synthesis of selenocysteine and its co-translational incorporation in selenoproteins in response to a UGA codon in frame require complex molecular machinery. In eukaryotes, components that participate in the reaction of selenocysteine formation were identified: SeryltRNA synthetase (SerRS), O-phosphoseryl-tRNA kinase (PSTK), SECIS Binding Protein 2 - SBP2, a selenocysteine-specific elongation factor (EFSec), selenophosphate synthetase 1 (SPS1) and selenophosphate synthetase 2 (SPS2), SEPSECS, SECp43 RNA binding protein, ribosomal protein L30, selenocysteine tRNA (tRNASec, SELC), and a specific sequence in the messenger RNA (SECIS element). The first step for selenocysteine incorporating is performed by SerRS that aminoacylates the tRNA with serine through serine activation by Mg2+ and ATP leading to the formation of an intermediate linked to the enzyme (Ser-AMP). Subsequently, the change of the Ser radical to tRNASec takes place followed by the enzymatic conversion of Ser-tRNASec to Sec-tRNASec. Through in silico analysis our group has identified components of the selenocysteine insertion machinery in species of Kinetoplastida. Homologues of tRNASec and the enzymes TbSerRS, TbSPS2, TbPSTK, TbSepSecS and TbEFSec were identified. Our main target is the structural study of the native SerRS from Trypanosoma brucei and SerRS in complex with the tRNASec and the tRNASer isoforms. A new methodology in the purification process of this enzyme has been developed, and through molecular exclusion chromatography, dynamic light scattering and analytical ultracentrifugation techniques we were able to determine the oligomeric state of TbSerRS. The result of dimers in solution corroborated with the data reported in the literature. Moreover, we were able to verify through studies of enzyme kinetics that the enzyme is active. The sedimentation equilibrium analytical ultracentrifugation technique also demonstrated the formation of the SerRS-tRNA complex, however, it did not allow the definition of the complex stoichiometry. Structural studies of the native enzyme and its interaction with SELC, tRNASer isoforms, L-serine, a non-hydrolyzable AMP analog, MgCl2, and smaller portions of tRNAs were performed by X-ray diffraction crystallography. Through this technique, seventeen data sets were collected, processed, and are being submitted to refinement processes. Initial structural analysis allowed the confirmation of the presence of two glycerol molecules in each monomer in the active site region in the native structure of TbSerRS and one dAMP molecule in the TbSerRS-dAMP complex.

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Barasa, Nathaniel Wafula. "Proteomic Characterization of Selenite Resistance in a strain of Enterobacter cloacae." Youngstown State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1221154755.

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Serrão, Vitor Hugo Balasco. "Caracterização das interações macromoleculares das proteínas envolvidas na síntese de selenocisteínas em Escherichia coli." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-10052017-081231/.

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O estudo de processos de tradução do código genético em proteínas desperta o interesse pelo seu papel central no metabolismo celular, em particular, o estudo da via de síntese de novos aminoácidos, como a selenocisteína e a pirrolisina, que resultam na expansão do código genético dos 20 aminoácidos canônicos para um total de 22 aminoácidos. A selenocisteína (Sec, U) é um aminoácido que representa a principal forma biológica do elemento selênio e sua incorporação ocorre através de um processo cotraducional em selenoproteínas como resposta ao códon UGA em fase, usualmente interpretado como códon de parada. Essa incorporação requer uma complexa maquinaria molecular distinta entre os três domínios da vida em que as selenoproteínas estão presentes: Bactéria, Arquéia e Eucária. Em Escherichia coli, a via se inicia com a aminoacilação do tRNA específico para a incorporação de selenocisteínas (SelC, tRNASec) com um resíduo de L-serina pela seril-tRNA sintetase (SerRS) formando o tRNA carregado Ser-tRNA[Ser]Sec que é entregue ao complexo homodecamérico selenocisteína sintase (SelA) responsável pela conversão Ser-Sec utilizando a forma biológica de selênio entregue pela enzima selenofosfato sintetase (SelD). Uma vez carregado com L-selenocisteína, o Sec-tRNASec é então carreado pelo fator de elongação específico para selenocisteínas (SelB) para a sua incorporação na cadeia polipeptídica nascente na posição UGA adjunta ao elemento SECIS (SElenoCysteine Insertion Sequence), uma estrutura em grampo presente no RNA mensageiro que indica o códon de inserção de selenocisteínas. Uma vez que elementos contendo selênio são tóxicos para o ambiente celular, interações entre as enzimas da via se fazem necessárias, onde as enzimas participantes em procariotos são conhecidas e caracterizadas individualmente, no entanto, suas interações macromoleculares nas diferentes etapas ainda não foram caracterizadas. Este projeto visa à caracterização macromolecular e estrutural das interações entre as enzimas SelA e SelB com os RNAs participantes tRNASec e SECIS além do ribossomo de E. coli. Para isso, amostras de SelA, SelB, tRNASec, SECIS e ribossomo foram obtidas através de diferentes metodologias. Para SelA e tRNASec foram utilizados protocolos já estabelecidos enquanto que, para SelB, fez-se necessário a otimização do protocolo previamente publicado e, consequentemente, nova caracterização biofísica através de metodologias como dicroísmo dircular (CD) e fluorescência intrínseca (IFS). Para análise das interações, medidas de espectroscopia de anisotropia de fluorescência (FAS), ultracentrifugação analítica (AUC) e calorimetria de varredura diferencial (DSC) foram utilizadas para determinação dos parâmetros de interação dos diferentes complexos estudados. Somado a isso, experimentos de cinética GTPásica foram realizados na formação dos complexos e, além disso, foram gerados modelos estruturais utilizando diferentes metodologias como espalhamento de raios-X a baixo ângulo (SAXS) além de estudos por microscopia eletrônica de transmissão (TEM). Os estudos propostos irão auxiliar no entendimento do mecanismo de incorporação deste aminoácido em bactérias bem como nos demais domínios da vida além de elucidar o mecanismo sequencial de eventos, provendo conhecimento e desenvolvendo metodologias para sistemas complexos de interação proteína-proteína e proteína-RNA. The study of genetic code processes in proteins is a central role in cell metabolism, in particular the study of the synthesis pathway of new amino acids, such as selenocysteine and pyrrolisine, which resulted in the expansion of the genetic code of the 20 canonical amino acids for 22 amino acids. Selenocysteine (Sec, U) is an amino acid that represents a major biological form of selenium element and its incorporation through a co-translational process in selenoproteins in response to the in-phase UGA-codon, usually interpreted as stop-codon. This incorporation requires a complex molecular machinery distinct between the three domains of life in which, as selenoprotein has present: Bacteria, Archaea and Eukaria. In Escherichia coli, an initiation pathway with an aminoacylation of the tRNA specific for the incorporation of selenocysteines (SelC, tRNASec) with an L-serine residue by seril-tRNA synthetase (SerRS) resulting in the charged tRNA Ser-tRNA[Ser] Sec that is delivered to the homodecameric complex selenocysteine synthase (SelA), responsible for Ser-Sec conversion using the biological form of selenium delivered by the enzyme selenophosphate synthetase (SelD). Once loaded with L-selenocysteine, Sec-tRNASec is then carried by the selenocysteine-specific elongation factor (SelB) for incorporation into the nascent polypeptide chain at the UGA position attached to the SECIS (SElenoCysteine Insertion Sequence) element, staple structure that indicates the insertion codon of selenocysteines. Since elements containing selenium are toxic to the cell, interactions between how pathway enzymes are made, where the enzymes participating in concepts are known and characterized individually, however, their macromolecular interactions in the different steps have not yet been characterized. This project aims at the macromolecular and structural characterization of the interactions between SelA and SelB enzymes with the RNAS tRNASec and SECIS participants in addition to the E. coli ribosome. For this, as samples of SelA, SelB, tRNASec, SECIS and ribosome were obtained through different methodologies. For SelA and tRNASec, protocols were used to determine parameters for SelB, it was necessary to optimize a previously published protocol and, consequently, a new biophysical characterization through methodologies such as circular dichroism (CD) and intrinsic fluorescence spectroscopy (IFS). To analyze the interactions, measurements of fluorescence anisotropy spectroscopy (FAS), analytical ultracentrifugation (AUC) and differential scanning calorimetry (DSC) were used to determine the interaction parameters of different complexes studied. In addition, GTPases activity experiments were carried out in the formation of the complexesand, in addition, we have generated models that characterize different methodologies such as small angles X-ray scattering (SAXS) and transmission electron microscopy (TEM). The proposed studies will aid in understanding the mechanism of incorporation of this amino acid into bacteria as well as the other domains of life besides elucidating the sequential mechanism of events, providing knowledge and development of methodologies for complex protein-protein and RNA-protein interaction systems.

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Cockman, Eric Michael. "Post-Transcriptional Regulation of Selenoprotein S." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1562593531805034.

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Koniev, Oleksandr. "Development of new bioselective ligation reactions." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF008/document.

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La ligation chimique implique la liaison des molécules de manière covalente pour former un nouveau complexe ayant les propriétés combinées de ses composants individuels. Ainsi, les composés naturels ou synthétiques avec leurs activités individuelles peuvent être conjuguer pour créer des substances possédant des caractéristiques uniques. Un domaine d' intérêt particulier à ces procédures est le marquage de protéines. Afin de simplifier et d'accélérer la découverte de nouvelles réactions de ligation bioselectives, nous avons conçu un système de screening rapide pour attribuer de la sélectivité et de la réactivité d'un groupement fonctionnel vers une série de dérivés d'acides aminés traçable. Une fonction chimique à propriétés prometteuse – 3-arylpropiolonitrile (APN) – a été identifiée. Les études comparatives ont démontré que cette technique offrait une meilleure sélectivité et stabilité par rapport à la technologie classique basée sur l’utilisation du groupement maléimide. L’utilisation de l’APN permet d’obtenir des bioconjugués propres et résistants à la décomposition, ce qui est d’une importance cruciale pour les applications médicales. Étude structure-réactivité nous a permis d'optimiser ses propriétés et de préparer une série de sondes fonctionnelles, dont un a été utilisé pour tester la sélectivité d'APN sur les mélanges modèles de peptides. De plus, les APN ont été trouvés à posséder une sélectivité élevée vers sélénocystéine: un acide aminé rare mais très important présent dans de nombreux enzymes actives. Une série des APN a été testée pour son activité inhibitrice envers une enzyme contenant sélénocystéine – thiorédoxine réductase – et s'est révélé posséder des activités élevées Enfin, une approche combinatoire de type split and mix a été développée visant à identifier des séquences peptidiques possédant la réactivité élevée avec les réactifs biosélectifs déjà connus Chemical ligation involves the linking of molecules in covalent manner to form a novel complex having the combined properties of its individual components. Thus, natural or synthetic compounds with their individual activities can be chemically combined to create unique substances possessing carefully engineered characteristics. A field of especial interest in such ligation procedures is protein labeling.To accelerate the discovery of new bioselective ligation reactions, we designed a screening system for fast assigning of the selectivity and reactivity of a given functional group owards series of UVGtraceable amino acid derivatives. As a result of our screening a promising cysteineGselective scaffold–3Garylpropiolonitrile (APN)–was identified. Its remarkable selectivity, high reactivity and of both starting and addition products in aqueous and organic media represents an important advantage compared to methodologies classically used for cysteine tagging. StructureGreactivity study allowed us to optimise its properties and toprepare a series of funcional probes, one of which was used for!an accurate test of APN selectivity on model mixtures of peptides. Furthermore, APN were found to possess an elevated selectivity towards selenocysteine:ararebut very important amino!acid found in many active enzymes.A series of APN was tested for their inhibitory activity towards one of such selenocysteineGcontaining enzyme–thioredoxine reductase–and was found to possess promising activities, which however still must be!optimised.Lastly, a screening system devoted to the discovery of reagents reactivity towards a sequence of amino acid residue was elaborated and allowed us to determine presumable discrepancy in reactivity of APN depending on the amino acid residue neighbouring the cysteine moiety. Such difference in reactivity may represent an important advantage for bioconjugation, and is currently under further investigation

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Papp, Laura V., and n/a. "Multiple Levels of Regulation of Human SECIS Binding Protein 2, SBP2." Griffith University. School of Biomolecular and Biomedical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20070208.145623.

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Selenium is an essential trace mineral of fundamental importance to human health. Its beneficial functions are largely attributed to its presence within a group of proteins named selenoproteins in the form of the amino acid selenocysteine (Sec). Recently, it was revealed that the human selenoproteome consists of 25 selenoproteins, and for many of them their function remains unknown. The most prominent known roles of selenoproteins are to maintain the intracellular redox homeostasis, redox regulation of intracellular signalling and thyroid hormone metabolism. Sec incorporation into selenoproteins employs a unique mechanism that involves decoding of the UGA stop codon. The process requires interplay between distinct, intrinsic features such as the Sec Insertion Sequence (SECIS) element, the tRNASec and multiple protein factors. The work presented in this thesis has focused on characterising the regulation of human SECIS binding protein 2, SBP2, a factor central to this process. Experimental approaches combined with bioinformatics analysis revealed that SBP2 is subjected to alternative splicing. A total of nine alternatively spliced transcripts appear to be expressed in cells, potentially encoding five different protein isoforms. The alternative splicing events are restricted to the 5?-region, which is proposed to be dispensable for Sec incorporation. One of the variants identified, contains a mitochondrial targeting sequence that was capable of targetting SBP2 into the mitochondrial compartment. This isoform also appears to be expressed endogenously within the mitochondria in cells. Previous reports have depicted SBP2 as a ribosomal protein, despite the presence of a putative Nuclear Localisation Signal (NLS). In this study it was found that SBP2 subcellular localisation is not restricted to ribosomes. Intrinsic functional NLS and Nuclear Export Signals (NESs), enable SBP2 to shuttle between the nucleus and the cytoplasm via the CRM1 pathway. In addition, the subcellular localisation of SBP2 appears to play an important role in regulating Sec incorporation into selenoproteins. The subcellular localisation of SBP2 is altered by conditions imposing oxidative stress. Several oxidising agents induce the nuclear accumulation of SBP2, which occurs via oxidation of cysteine residues within a novel redox-sensitive cysteine rich domain (CRD). Cysteine residues were to form disulfide bonds and glutathione-mixed disulfides during oxidising conditions, which are efficiently reversed in vitro by the thioredoxin and glutaredoxin systems, respectively. These modifications negatively regulate selenoprotein synthesis. Cells depleted of SBP2 are more sensitive to oxidative stress than control cells, which correlated with a substantial decrease in selenoprotein synthesis after treatment with oxidising agents. These results provide direct evidence that SBP2 is required for Sec incorporation in vivo and suggest that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins. Collectively, these results suggest that SBP2 is regulated at multiple levels: by alternative splicing, changes in subcellar localisation and redox control.

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Fernandes, Adriano de Freitas. "Análise da especificidade do tRNASec entre o fator de elongação específico para selenocisteínas (SelB) e Seril-tRNA Sintetase (SerRS) de Escherichia coli." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-18052017-081552/.

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A selenocisteína (Sec, U) é o aminoácido que representa a principal forma biológica do elemento selênio e sua incorporação é um processo co-traducional em selenoproteínas como resposta ao códon UGA em fase e requer uma complexa maquinaria molecular. O repertório completo de genes envolvidos nessa via de síntese em procariotos é conhecido, porém algumas das interações moleculares ainda não foram totalmente esclarecidas. Este projeto visa à caracterização molecular nas interações entre o Fator de Elongação específico para incorporação de Sec (SelB) e Seril-tRNA sintetase (SerRS) com distintas construções do tRNASec de Escherichia coli afim de compreender a sua especificidade, seletividade e ordem de eventos. Para isso, medidas de Espectroscopia de Anisotropia de Fluorescência (FAS), Ultracentrifugação Analítica (AUC) e Calorimetria de Varredura Diferencial (DSC) foram utilizadas para determinação das constantes de interação desses complexos proteína-tRNA. Além disto, experimentos de Espalhamento de Raios-X a baixo ângulo (SAXS) e Microscopia eletrônica de transmissão por contraste negativo (NS-EM) foram realizados para elucidação estrutural destes complexos. Os estudos propostos irão auxiliar no entendimento do mecanismo de incorporação e de especificidade do tRNA para este aminoácido em bactérias bem como nos demais domínios da vida além de possibilitar um aumento na compreensão de complexos do tipo proteína-tRNA bem como salientar a importância dos elementos estruturais do tRNA para sua especificidade no processo de síntese de novas proteínas. Selenocysteine (Sec, U) is an amino acid that represents the main biological form of the selenium element and its incorporation is a co-translational process in selenoproteins in response to the in-phase UGA codon and requires complex molecular machinery. The complete repertoire of genes involved in this pathway of synthesis in prokaryotes is known, although some of the molecular interactions have not yet been fully elucidated. This project aims at the molecular characterization in the interactions between the specific elongation factor for the incorporation of Sec (SelB) and Seril-tRNA synthase (SerRS) with different constructions of tRNASec from Escherichia coli in order to their specificity, selectivity and order of events. For this, measurements using Fluorescence Anisotropy Spectroscopy (FAS), Analytical Ultracentrifugation (AUC) and Differential Scanning Calorimetry (DSC) were employed to determine the interaction constants of the protein-tRNA complexes. In addition, Small Angle X-Ray Scattering (SAXS) experiments and negative stain transmission electron microscopy (NS-EM) were performed for structural elucidation of these complexes. The proposed studies will help to understand the mechanism of tRNA incorporation and specificity for this amino acid in bacteria as well as other domains of life. In addition, it allows an increase in the understanding of protein-tRNA-like complexes as well as emphasizing the importance of structural elements of tRNA for its specificity in the process of synthesis of new proteins.

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Costa, Fernanda Cristina. "Validação da via de biossíntese de selenocisteína e selenoproteínas em Trypanosoma por RNA de interferência." Universidade Federal de São Carlos, 2012. https://repositorio.ufscar.br/handle/ufscar/5398.

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Made available in DSpace on 2016-06-02T20:20:33Z (GMT). No. of bitstreams: 1 4383.pdf: 5759644 bytes, checksum: 0a6a6bb722062f7934be619267686311 (MD5) Previous issue date: 2012-04-24 Universidade Federal de Minas Gerais Selenium (Se) is an essential element found in selenoproteins as the 21st amino acid (Selenocysteine Sec).For the Sec incorporation and the related biosynthetic pathaway, several elements are required: tRNASec, a UGA codon and a Sec insertion sequence (SECIS), a conserved motif downstream of the selenoprotein encoding gene. Selenoproteins generally participate in the cellular redox balance, playing an important role on cell growth and proliferation. These proteins, as well as the the Sec synthesis pathway, are present in members of the Bacteria, Archaea and Eukarya domains, being identified in several protozoa, including the kinetoplastids. Auranofin, a gold-contaning antirheumatic drug, is a known selenoproteins inhibitorand Trypanosoma brucei and Leishmania majorcells are sensitive to this compound, with a LD50 in nanomolar range. This indicates a possible dependence of these parasites on selenoproteins. Theselenophosphate synthetase (SELD/SPS2) is responsible for the formation of monoselenophosphate from selenide and ATP, being essencial for selenoprotein biosynthesis. SPS2 knockdown led to apoptosis under sub-optimal growth conditions. The selenoproteome of these flagellated protozoa consists of distant homologs of the mammalian SelK and SelT, and a novel selenoprotein designated SelTryp, a kinetoplastidspecific protein. The functions of any of these selenoproteins are not known.We have investigated the effect of their downregulation in T. brucei to interpret their possible physiological role. The TbSelK depletion shows no effect on growth under optimal conditions, but the cells became more sensitive to endoplasmic reticulum stress agents and oxidative stress, suggesting that SelK is an ER stress-regulated protein and plays an important role in protecting T. brucei cells from ER stress agent. The TbSelT gene silence by RNA interference hampers the parasite survival, but the sensitivity to the agents tested was not asevident as it was forTbSelK, suggesting a role for TbSelT in protection against stress, but not specifically ER stress. Our results show the importance of selenocysteine and selenoproteins to parasite survival. Selênio (Se) é um elemento essencial encontrado em selenoproteínas na forma do 21º aminoácido selenocisteína (Sec U). A incorporação co-traducional de Sec depende de uma complexa via de síntese, de um códon de terminação UGA em fase de leitura e uma estrutura terciária do RNA mensageiro conhecida como elemento SECIS. A maioria das selenoproteínas conhecidas participa de processos de manutenção do estado redox das células, tendo um importante papel no crescimento e proliferação celular. Essas proteínas, bem como os componentes da via de síntese de Sec, estão presentes em membros dos domínios de Bactérias, Arquéais e Eucaria, tendo sido identificada em diversos protozoários, incluindo os kinetoplastidas. Auranofin, um composto de ouro usado como agente antireumático, tem sido descrito como um inibidor de selenoproteínas através de sua ligação com o aminoácido selenocisteína e células de Trypanosoma brucei e Leishmania major são altamente sensíveis a este composto, apresentando um LD50 na faixa de nanomolar. Esta evidência indica uma possível dependência destes parasitas por selenoproteínas e consequentemente pela sua via de síntese. A selenofosfato sinetase (SELD/SPS2) é a enzima responsável pela síntese de monoselenofosfato a partir de seleneto e ATP, sendo, portanto uma proteína fundamental na síntese de selenocisteína. Sua depleção levou a apoptose celular quando mantidas em condições de estresse. Esse efeito pode ser causado pela consequente falta das selenoproteínas ou pelo acúmulo de espécies tóxicas de selênio, como o seleneto. Os protozoários apresentam número reduzido de selenoproteínas e kinetoplastidas apresentam 3, duas homólogas distantes de mamíferos, SelK e SelT, e uma nova proteína exclusiva denominada SelTryp, que não apresentam homologia com nenhuma outra proteína descrita. O papel dessas proteínas não é conhecido, e nós investigamos suas possíveis funções através da inibição de sua expressão. A depleção de TbSelK não mostrou efeito sob condições normais, mas tornou as células mais sensíveis a agentes indutores de estresse de retículo endoplasmático, o que nos permite inferir uma função de manutenção da homeostase dessa organela. A depleção de TbSelT causou uma diminuição no crescimento celular, mas o aumento da sensibilidade aos agentes indutores de estresse não foi tão pronunciada como em TbSelK. Nossos resultados revelam a importância de selenocisteína para parasitas, uma vez que esses organismos enfrentam diversos tipos de estresses para manter a viabilidade e a progressão da doença nos diferentes hábitats encontrados ao longo do seu ciclo de vida.

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Tavares, Kaio Cesar Simiano. "Biossíntese de selenocisteína em Trypanosoma evansi." Universidade do Estado de Santa Catarina, 2011. http://tede.udesc.br/handle/handle/847.

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Made available in DSpace on 2016-12-08T16:24:10Z (GMT). No. of bitstreams: 1 PGCA11MA081.pdf: 22717 bytes, checksum: 41112e9e69fe9ceac2afe9b11e56e60f (MD5) Previous issue date: 2011-02-25 Trypanosoma evansi is the pathogenic trypanosomatid with the worldwidest distribution, generating economic losses in Africa, South America, Europe, Asia and Oceania. This protozoan is the etiologic agent of the disease know as Surra or Mal das Cadeiras, wich affects almost all species of mammals, with a recent case in humans. An important metabolic pathway described in all the three kingdoms of life is the incorporation of selenium into proteins, wich mainly has an antioxidant function. Selenium is used in the form of the amino acid selenocysteine, which is incorporated into the nascent polypeptide co-translationally through the stop codon "UGA". Some elements plays a key role into this pathway: a signaling nucleotide structure in the messenger RNA (SECIS), a specifc tRNA (tRNASec) and an enzyme complex that allows the conversion of selenium in its active form monoselenophosphate (SPS), its aminoacylation in tRNASec (SerRS, PSTK, SepSecS) and the coupling of nucleotidic and proteic structures (SECIS, EF-Sec, SBP2) in the UGA codon to translation and insertion of selenocysteine into the protein. This work demonstrated that T. evansi express the genes selB (EF-Sec), selC (tRNASec), selD (SPS) and pstk in its mRNA. The domains analysis of T. evansi selB, selD and PSTK genes found regions that are consistent with the predicted proteins functions. The predicted secondary structure of T. evansi tRNASec shares the most of the characteristics of eukaryotic tRNASec. Using Southern Blot, we showed that selB, selD and pstk are single copie genes in T. evansi genomic DNA. The SPS proteis was correctly localized in the total protein extract of the parasite, with a 43 kDa band. The same protein has a cytoplasmatic localization in T. evansi, as showed by indirect immunofluorescence. The gene of a trypanosomatid exclusive selenoprotein, selTRYP, was amplified of the cDNA and sequenced. Through these results, we suggest that T. evansi is capable of using selenium for the formation of selenoproteins, and the presence of the selTRYP, selb, selc, seld and pstk genes may indicate a potential future therapeutic target, since recent data show an increase in the parasite resistance to the commercial available drugs in different continents O Trypanosoma evansi é o tripanossomatídeo patogênico de maior distribuição mundial, causador de prejuízos econômicos na África, América do Sul, Europa, Ásia e Oceania. Este protozoário é o agente etiológico da doença conhecida como Surra ou Mal das Cadeiras, que afeta praticamente todas as espécies de mamíferos, com um recente caso em humanos. Uma importante via metabólica descrita em todos os reinos da vida é a incorporação de selênio em proteínas, com função, principalmente, antioxidante. O selênio é utilizado na forma do aminoácido selenocisteína, que é incorporada ao polipeptídeo nascente co-traducionalmente através do códon de parada UGA . Para que isto ocorra, são necessárias uma estrutura nucleotídica sinalizadora no RNA mensageiro (SECIS), um RNA transportador específico (tRNASec) e um complexo de enzimas que permitem a conversão do selênio em sua forma ativa, monoselenofosfato (SPS), sua aminoacilação no tRNASec (SerRS, PSTK, SepSecS) e o acoplamento de estruturas nucleotídicas e protéicas (SECIS, EF-Sec, SBP2) para que o códon UGA seja traduzido em selenocisteína e a mesma seja inserida na proteína. Neste trabalho foi demonstrado que o T. evansi expressa os genes selB (EF-Sec), selC (tRNASec), selD (SPS) e PSTK. A análise de domínios dos genes selB, selD e PSTK de T. evansi encontrou regiões condizentes com as características funcionais das proteínas formadas. A estrutura secundária predita do tRNASec de T. evansi compartilha a maioria das características dos tRNASec de eucariotos. Através da técnica de Southern Blot, demonstrou-se que os genes selB, selD e PSTK possuem cópia única no DNA genômico de T. evansi. Utilizando-se Western Blot, a proteína SPS foi localizada corretamente no extrato protéico do protozoário, formando uma banda de 43 kDa. Foi realizada também uma imunolocalização da SPS, sendo que a mesma possui localização citoplasmática neste protozoário. O gene de uma selenoproteína exclusiva de tripanossomatídeos, a selTRYP, foi amplificado do cDNA e parcialmente sequenciado. Através desses resultados, sugere-se que o T. evansi é capaz de utilizar selênio para a formação de selenoproteínas, e a presença dos genes da via de inserção de selenocisteína pode indicar um potencial futuro alvo terapêutico, visto que recentes dados demonstram um crescimento de cepas resistentes aos medicamentos disponíveis no mercado em vários continentes

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Mariotti, Marco. "Computational genomics of selenoproteins." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/295583.

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Selenoproteins are a diverse class of proteins containing selenocysteine, the 21st aminoacid. Selenocysteine is inserted co-translationally, recoding very specific UGA codons through a dedicated machinery. Standard gene prediction programs consider UGA only as translational stop, and for this reason selenoprotein genes are typically misannotated. In the past years, we developed computational tools to predict selenoproteins at genomics scale. With these, we characterized the set of selenoproteins across many sequenced genomes, and we inferred their phylogenetic history. We dedicated particular attention to selenophosphate synthetase, a selenoprotein family required for selenocysteine biosynthesis, that can be used as marker of the selenocysteine coding trait. We show that selenoproteins went through a very diverse evolution in different lineages. While very conserved in vertebrates, selenoproteins were lost independently in many other organisms. Using genome sequencing, we traced with precision the path of genomic events that lead to recent selenoprotein extinctions in certain fruit flies. Les selenoproteïnes s’agrupen en una classe heterogènia de proteïnes les quals contenen selenocysteïna, l’aminoàcid 21. La selenocisteïna és insertada durant el procés de traducció, recodificant codons UGA molt específics, mitjançant una maquinàiria dedicada. Els programes estàndard de predicció de gens interpreten el codó UGA només com a senyal d’stop de la traducció, i per aquesta raó els gens de selenoproteïness solen estar mal anotats. En els darrers anys, hem desenvolupat eines computacionals per a predir selenoproteïnes a escala genòmica. Amb aquestes, hem caracteritzat el conjunt de selenoproteïnes en aquells genomes que han estat seqüenciats, inferint la seva història filogenèitca. Hem dedicat especial ateníció a la família selenophosphate synthetase, selenoproteïna necessària per a la síntesi de selenocisteïna, i que per tant pot ser utilitzada com a marcador de codificació de selenocisteïna Mostrem que les selenoproteïnes han patit una evolució molt diversa en diferents llinatges. Tot i que es troben molt conservades en vertebrats, les selenoproteïnes van ser perdudes de manera independent en molts altres organismes. Gràcies a la sequenciació de genomes, vam traçar amb precisió els esdeveniment que van portar a l’extinció de selenoproteïnes a diverses espècies de drosòfila.

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Alsharif, Ifat. "Etudes in vitro et in vivo de l'effet neuroprotecteur d'un peptide dérivé de la sélénoprotéine T, le PSELT, dans un modèle de la maladie de Parkinson." Thesis, Normandie, 2018. http://www.theses.fr/2018NORMR010/document.

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Les maladies neurodégénératives telles que la maladie d'Alzheimer, la maladie de Parkinson et la maladie de Huntington sont des pathologies progressives qui affectent le système nerveux, conduisant à la mort de certaines cellules nerveuses. Toutes ces maladies se caractérisent par la perte progressive de neurones dans des régions plus ou moins localisées du système nerveux, entraînant des complications cognitives, motrices ou perceptives. La MP est caractérisée par une dégénérescence sélective et progressive des neurones dopaminergiques situés dans la substance noire pars compacta (SNc), et de leurs terminaisons nerveuses qui normalementlibèrent la dopamine dans le striatum. Bien que les causes exactes de la MP soient inconnues, de nombreuses études ont démontré le rôle important du stress oxydatif dans la dégénérescence des neurones dopaminergiques. D’ailleurs, un niveau élevé de radicaux libres est observé dans le cerveau de patients post-mortem. Ces observations suggèrent que les protéines qui jouent un rôle dans la protection des neurones contre les effets du stress oxydatif peuvent représenter des cibles thérapeutiques intéressantes. En effet, pour maintenir l'équilibre d'oxydo-réduction, les cellules recrutent plusieurs enzymes réductrices dont des membres de la famille des sélénoprotéines. Des résultats obtenus dans notre laboratoire ont montré que la sélénoprotéine T (SelT), une nouvelle sélénoprotéine identifiée dans les cellules nerveuses dans notre laboratoire, est fortement exprimée dans les conditions de dégénérescence des neurones suite à un stress oxydant, et exerce un rôle neuroprotecteur. Ce rôle est assuré par son site actif contenant une cystéine et une sélénocystéine. Le but de ce travail de thèse était de valider l’utilisation d’un peptide nommé PSELT contenant le coeur actif de la SelT en tant que traitement neuroprotecteur dans la MP. Le traitement des cellules de neuroblastome SH-SY5Ypar le peptide PSELT réduit significativement les niveaux des radicaux libres et stimule la survie cellulaire en inhibant l’apoptose. Le PSELT semble traverser la membrane plasmique pour exercer son effet. In vivo, l’administration intranasale du PSELT protège les neurones et les fibres dopaminergiques dans un modèle de la MP chez la souris traitée par le 1-méthyl-4-phényl-1,2,3,6-tétrahydropyridine (MPTP). Le peptide PSELT augmente le taux de la tyrosine hydroxylase et inhibe l’apoptose, ce qui aboutit à une amélioration des troubles moteurs induits par le MPTP chez les animaux. L’ensemble de ces résultats montrent pour la première fois qu’un peptide issu de la SELT, le PSELT, est capable de protéger les neurones dopaminergiquesin vitro et in vivo et d’améliorer l’activité motrice des animaux modèles de la MP, ouvrant la voie au développement d’une nouvelle thérapie de neuroprotection pour la MP Résumé en anglais non fourni

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Godin, Simon Michel Dominique. "Impact et potentiel d’une supplémentation en sélénium des aliments piscicoles : apport de la spéciation." Thesis, Pau, 2015. http://www.theses.fr/2015PAUU3050.

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Cette thèse a pour but de déterminer les espèces séléniées (spéciation) issues du métabolisme du poisson (truite) suite à l’enrichissement en sélénium, organique ou inorganique, des aliments piscicoles. Ces informations, complémentaires à celles utilisées en nutrition, sont nécessaires pour juger de la nécessité et de l’adéquation d’une supplémentation en sélénium des aliments végétaux afin de garantir les fonctions biologiques dépendantes de Se, ainsi que la qualité nutritionnelle des poissons. δa capacité équivalente d’un apport en sélénium inorganique ou organique à relever les niveaux de sélénoprotéines en cas de carence a ainsi été mise en évidence, ce qui diffère des conclusions habituellement obtenues sur la base de mesures du sélénium total. δ’utilisation de traceurs mono-spécifiques et mono-isotopiqueslors de la préparation d’échantillons (sang/plasma de truite) a montré l’existence de réactions co-précipitation et/ou d’interactions entre analytes séléniés et protéines démontrant l’attention particulière qui doit être portée à l’étape de préparation d’échantillon This PhD aims at the determination of selenium species (speciation) of fish metabolism after inorganic or organic selenium enrichment of aquaculture feeds. This information, complementary to the one obtained in the nutrition field, is necessary to assess the requirement and the suitability of the selenium supplementation of plant based feed in order toensure selenium dependent biological functions, as well as the nutritional quality of fish. The equivalent ability of inorganic and organic selenium to raise selenoproteins levels in case of deficiency was revealed, which differs with conclusion usually obtained based on total selenium measurements. The use of monospecific and monoisotopic tracers during sample preparation (trout plasma/blood) showed the existence of co-precipitation reactions and interactions between selenized analytes and proteins demonstrating thus that attention has to be paid to the sample preparation step

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Bianga, Juliusz. "Développement d’une approche analytique pour la caractérisation du sélénoprotéome in vivo." Thesis, Pau, 2013. http://www.theses.fr/2013PAUU3003/document.

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Le sélénium est un micronutriment essentiel pour des nombreux organismes vivants, y compris l’homme. Son rôle est lié à sa présence dans des sélénoprotéines sous forme d’un acide aminé, génétiquement encodé – la sélénocystéine. Il y a 25 sélénoprotéines encodées dans le génome humain. Leurs fonctions, la cinétique et la hiérarchie d'expression se trouvent au cœur des problématiques de recherche concernant le sélénium et la santé humaine. Il existe également un autre type de protéines où le sélénium est inséré par un remplacement partiel du soufre dans la méthionine mais aussi, potentiellement, dans la cystéine. Ces protéines suscitent l’intérêt dans les sciences de nutrition comme source de sélénium biodisponible dans l’alimentation naturelle et supplémentée. L'objectif de cette thèse a été la mise au point de méthodologies analytiques visant la spéciation du sélénium incorporé dans les protéines à l’échelle du protéome entier. Une procédure inédite a été développée pour la détection globale de protéines séléniées dans des gels d’électrophorèse bidimensionnelle par l’imagerie d’ablation laser ICP MS (spectrométrie de masse plasma à couplage inductif) permettant de s’affranchir de l’utilisation de l’isotope radioactif 75Se. Les autres avancées comprennent la mise en place d’un couplage robuste de HPLC capillaire avec l’ICP MS pour la détection des sélénopeptides dans des microvolumes de digestats trypsiques des protéines extraites du gel ainsi que la mise en place des protocoles d’identification des protéines séléniées par la spectrométrie de masse électrospray en tandem utilisant la trappe orbitale (Orbitrap). Les méthodes développées ont permis (i) la caractérisation de la part du protéome sélénié contenant la sélénocystéine chez la levure séléniée, (ii) l’identification des protéines majeures qui accumulent le sélénium dans le blé, et (iii) le dosage semi quantitatif et la caractérisation globale des sélénoprotéomes (GPx1, GPx4, TRxR1, TRxR2, Sel15kDa) dans les lignées cellulaires Selenium is an essential micronutrient for many living organisms including man. Its role is related to selenoproteins which contain genetically encoded selenocysteine. There are 25 selenoproteins encoded in the human genome. Their function, expression kinetics and hierarchy have been a topic of intense research in life sciences. There is another type of proteins which contain selenium inserted non-specifically by partly replacing sulphur in methionine and, potentially, cysteine. They are of interest in nutrition science as source of bio-available selenium in natural and supplemented foods. The goal of this Ph.D. was the development of methodologies for the analysis of selenium-containing proteins on the entire proteome scale. A novel procedure was developed for their global detection in 2D electrophoretic gels par laser ablation inductively coupled plasma mass spectrometry (ICP MS) imaging permitting to avoid the use of the radioactive 75Se. The other developments included (i) a robust capillary HPLC – ICP MS coupling allowing the detection of Se-containing peptides in microliter volumes of the digests of proteins extracted from the gel and (ii) protocols allowing the targeted identification of the Se-containing proteins by a parallel capillary HPLC - electrospray Orbitrap MS/MS. The methods developed allowed (i) the characterisation of the selenocystein-containing part of the selenoproteome of Se-enriched yeast, (ii) identification of the major Se-accumulating proteins in wheat, and (iii) semiquatitive analysis and global identification of the selenoproteomes (GPx1, GPx4, TRxR1, TRxR2, Sel15kDa) expressed in different human cell lines

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Parther, Tina [Verfasser]. "Die Peroxidase-Aktivität Selenocystein-haltiger Proteine des strikt anaeroben Bakteriums Eubacterium acidaminophilum / von Tina Parther." 2003. http://d-nb.info/96943233X/34.

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Gursinsky, Torsten [Verfasser]. "Selenoprotein-codierende mRNAs aus Eubacterium acidaminophilum : Erkennung durch den Selenocystein-spezifischen Elongationsfaktor SelB und Translation in Escherichia coli / von Torsten Gursinsky." 2002. http://d-nb.info/967124549/34.

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Ganichkin, Oleg. "Crystal structure analysis of selenocysteine biosynthesis components." Doctoral thesis, 2010. http://hdl.handle.net/11858/00-1735-0000-0006-B69B-9.

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Kotini, Suresh Babu. "Molecular mechanism of selenocysteine incorporation in bacterial translation." Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-000D-F0BB-9.

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Dissertations / Theses on the topic 'Selenocystein'

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