A Dissulfeto Isomerase Proteína (PDI) é uma chaperona redox essencial responsável pela inserção correta das ligações dissulfeto em proteínas nascentes no retículo endoplasmático. Nesta localização celular, bem como em outras regiões, como na superfície celular, a PDI atua na manutenção da homeostase redox e sinalização. Houve substanciosa evolução no conhecimento sobre a estrutura e funções da PDI, graças a estudos in vitro que utilizam a PDI purificada, quimeras ou seus domínios isolados. Nestas abordagens experimentais, as medidas das atividades redutase e chaperona da PDI são realizadas de forma relativamente simples. Entretanto, medir a atividade isomerase, que é a atividade autêntica da família das PDIs, é tecnicamente bastante complexo. Em células e tecidos, o papel da PDI tem sido descrito com base principalmente em estratégias experimentais de ganho e perda de função. Todavia, ainda há pouca informação na correlação entre os resultados funcionais com a medida das atividades da PDI. Este trabalho compila os principais métodos descritos para medir as quatro atividades da PDI: tiol redutase, tiol oxidase, tiol isomerase e chaperona, com ênfase na descrição de controles e interferentes críticos, como os tampões que contém surfactantes. Ainda, discutir-se-á criticamente os resultados obtidos quando da transposição destes métodos para amostras de homogenatos (celular ou tecidual) / Protein disulfide isomerase is an essential redox chaperone from endoplasmic reticulum, responsible for correct disulfide bond insertion in nascent proteins. At the endoplasmic reticulum and other locations including the cell surface, PDI accounts for redox homeostasis and signaling. Knowledge about PDI structure and function evolved substantially from in vitro studies using purified PDI and chimeras. In these experimental scenarios, PDI reductase and chaperone are readily approachable. However, isomerase activity, the hallmark of PDI family, is significantly complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function experiments. However, there is limited information regarding correlation of these results with PDI activities. In this manuscript, we put together the main methods described for measuring the four PDI activities: thiol reductase, thiol oxidase, thiol isomerase and chaperone, with emphasis on controls and critical interferents, such as detergent-containing buffers. We also discuss the transposition of these methods from purified PDI to cellular or in vivo samples, with critical thoughts about the interpretation of results

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Abstract THE CHARACTERIZATION OF CHIMERIC CHAPERONE FLAGRP170 AS A NOVEL RADIOPROTECTANT By Tyler Nguyen, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2017 Major Director: Dr. Xiang-Yang (Shawn) Wang, Ph.D., Professor, Department of Human and Molecular Genetics Radiation therapy (RT) is restricted by toxic effects on adjacent normal tissue, which limits RT efficacy in cancer treatment. Damage to normal tissue, such as radiosensitive intestine and bone marrow compartments, results in acute radiation damage. To reduce normal tissue injury in the setting of RT, we examine the potential radioprotectant, Flagrp170, a chimeric protein. Flagrp170 is comprised of glucose-regulated protein-170 (Grp170) and a NF-κB activating sequence derived from flagellin. We show that Flagrp170 can protect normal tissues post irradiation, indicated by TUNEL and clonogenic assays. However, treatment with Flagrp170 does not influence tumor response to RT. Studies indicate that Flagrp170 activates the transcription factor NF-κB, a strong pro-survival signal. In addition, Flagrp170 can induce production of radioprotective cytokines as well. Data suggests that Flagrp170 has potential as a novel radioprotectant in the setting of RT. The combination of Flagrp170 therapy and RT may lead to improved treatment outcomes.

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L’Autophagie médiée par les protéines chaperonnes (ou CMA pour Chaperone-Mediated Autophagy) est une voie majeure du catabolisme lysosomal considérée aujourd’hui comme un acteur central de contrôle de nombreuses fonctions cellulaires, et dont les défauts sont associés à plusieurs pathologies humaines, dont des maladies neurodégénératives, des cancers et des troubles du système immunitaire. Selon l’idée actuellement admise, cette fonction cellulaire n’existerait que chez les mammifères ou les oiseaux, qui seraient les seuls à exprimer la protéine LAMP2A, une protéine nécessaire au fonctionnement de la CMA. Or, récemment, nous avons pu mettre en évidence l’existence de séquences exprimées présentant une forte homologie avec LAMP2A de mammifères chez plusieurs espèces de poissons, remettant ainsi en question ce point de vue et suggérant que la CMA soit apparue beaucoup plus tôt au cours de l'évolution qu'on ne l'avait initialement cru. Dans cette thèse, nous retraçons l’histoire évolutive du gène LAMP2 chez les vertébrés. Nous démontrons que ce gène est apparu après la seconde duplication complète du génome survenue chez l'ancêtre commun des vertébrés il y a environ 500 millions d'années. En outre, en adaptant une méthode récemment décrite pour mesurer l’activité de la CMA dans des cellules de mammifères à une lignée de fibroblastes de medaka (Oryzias latipes), nous apportons la preuve de l’existence de cette fonction cellulaire chez cette espèce de poisson. Enfin, afin de caractériser le rôle physiologique de cette fonction chez les poissons, nous avons procédé à l’invalidation par crispR-cas9 de lamp2a chez le medaka. Les poissons générés présentaient de sévères perturbations du métabolisme intermédiaire, comme précédemment décrit chez des souris dont LAMP2A a été invalidée dans le foie. Dans l’ensemble, ces résultats démontrent clairement, et pour la toute première fois, qu’il existe bien une activité CMA fonctionnelle chez les poissons, et apportent ainsi de nouvelles perspectives dans ce domaine de recherche, notamment en autorisant l'utilisation de modèles génétiques complémentaires, tels que le poisson zèbre ou le medaka, pour faire avancer nos connaissances sur les mécanismes régissant cette fonction cellulaire. / Chaperone-Mediated Autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player in the control of numerous cellular functions, and whose defects have been associated to several human pathologies, including neurodegenerative diseases, cancers and immune disorders. To date, this cellular function was presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in non-tetrapod species. However, we recently identified the existence of expressed sequences displaying high homology with the mammalian LAMP2A in several fish species, challenging that view and suggesting that CMA appeared much earlier during evolution than initially thought. In the present thesis, we first present new evidences about the evolutionary history of the gene LAMP2 in vertebrates. We demonstrate that LAMP2 appeared after the second whole genome duplication that occurred at the root of the vertebrate lineage approximately 500 million years ago. By using a fluorescent reporter previously used to track CMA in mammalian cells, we then revealed the existence of a CMA-like pathway in a fibroblast cell line of the fish medaka (Oryzias latipes). Finally, to address the physiological role of Lamp2a in fish, we generated, medaka knockout for the splice variant lamp2a, and found severe alterations in the intermediary metabolism, as previously demonstrated in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22293号 / 理博第4607号 / 新制||理||1661(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 細川 暢子, 教授 森 和俊, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Fabry disease is an inherited X-linked recessive disorder caused by mutations in the galactosidase alpha (GLA) gene, leading to deficiencies in α-galactosidase A (α-GAL) enzyme production. α-GAL, a lysosomal glycosidase, catalyzes the removal of a terminal α-galactose; however, loss of α-GAL activity leads to accumulation of globotriaosylceramide (an endogenous substrate) and the eventual onset of the disease. Approved treatments for Fabry disease include enzyme replacement therapy and pharmacological chaperone therapy. In the latter treatment, 1-deoxygalactonojirimycin (DGJ), a pharmacological chaperone, is administered to Fabry disease patients, leading to increased enzymatic activity. The DGJ iminosugar acts as a competitive inhibitor of α-GAL, and upon addition at sub-inhibitory concentrations, the α-GAL activity in the cell increases. At pH 7.5, the DGJ binds and stabilizes both wild type and mutant α-GAL and can thus drive the folding of the α-GAL protein (Guce 2011). DGJ has been clinically approved to treat a subset of the more than 900 known mutations in the GLA gene. These approvals come from the chaperone activity data published by Amicus Therapeutics (Benjamin 2017). However, these assays cost money, time, and effort to perform, and novel mutations are discovered annually. Using molecular dynamics energy calculations in the Schrödinger software package, we developed a model to predict successful chaperoning of the mutants. Overall, the results are directly applicable to Fabry disease, but could also be applied to the much larger family of protein folding diseases, including Alzheimer's, Parkinson's and Huntington's diseases.

Le cancer de la prostate est le second cancer diagnostiqué chez les hommes dans le monde. Malgré le développement de nouveaux traitements au cours de ces cinq dernières années, les chimiothérapies par taxanes, docetaxel et cabazitaxel, restent des traitements de référence dans la prise en charge des patients atteints de cancer de la prostate métastatique résistant à la castration. Cependant, des résistances primaires et acquises émergent chez environ la moitié des patients. C’est pourquoi, il est urgent de découvrir et de comprendre les mécanismes de résistance aux taxanes afin d’identifier de nouvelles cibles thérapeutiques. En effet, de nouvelles thérapies ciblées peuvent émerger de la compréhension des voies de signalisation impliquées dans le cancer de la prostate pour contourner la chimiorésistance et améliorer les traitements. Les protéines chaperonnes jouent un rôle clef dans la régulation de l’homéostasie cellulaire et dans le développement de résistance aux traitements. Elles constituent donc des cibles thérapeutiques potentielles pour contourner la chimiorésistance. En réalisant un criblage fonctionnel par siARN à partir de profils d’expression génique, nous avons identifié FKBP7, une chaperonne moléculaire encore jamais étudiée chez l’homme, impliquée dans la résistance au docetaxel et au cabazitaxel. FKBP7 est surexprimée dans les tumeurs de la prostate et son expression est corrélée avec la récurrence chez les patients ayant reçu du docetaxel en thérapie néoadjuvante. De plus, FKBP7 est surexprimée dans des lignées cancéreuses prostatiques résistantes aux taxanes et son expression est nécessaire à leur croissance in vitro et à la croissance tumorale dans un modèle murin de résistance au docetaxel. Par des approches de protéomique haut-débit, nous avons identifié la voie de signalisation régulée par FKBP7 qui est responsable de la survie des cellules chimiorésistantes. Enfin, nous proposons une stratégie thérapeutique pour contourner la chimiorésistance au docetaxel et au cabazitaxel en ciblant l’effecteur moléculaire en aval de FKBP7. / Prostate cancer is the second cancer diagnosed among men worldwide. Beside approval of new therapies in the last five years, chemotherapeutic agents, docetaxel and cabazitaxel taxanes remain key treatments for metastatic castration resistant prostate cancers. However, primary and acquired resistance to taxanes still emerged in about half of patients. There is therefore an urgent need to discover and understand the taxane resistance mechanisms in order to identify new therapeutic targets. Indeed, targeted therapies that exploit the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes. Molecular chaperones play a key role in the regulation of cellular homeostasis and the development of treatment resistance, and are promising therapeutic targets. Using high throughput siRNA functional screening based on a gene expression signature, we identified FKBP7, involved in acquired resistance to docetaxel and cabazitaxel. FKBP7 is a molecular chaperone that has not been studied in human so far. FKBP7 is overexpressed in prostate tumors and its expression is correlated with recurrence in patients who received docetaxel as neoadjuvant therapy. Moreover, FKBP7 is upregulated in taxane resistant prostate cancer cell lines and its expression sustains their growth in vitro and in a mice model of Docetaxel resistance. Using a high throughput proteomic approach, we identified the signaling pathway regulated by FKBP7 which is responsible for the survival of chemoresistant cells. Finally, we proposed a promising therapeutic strategy to overcome both docetaxel and cabazitaxel chemoresistance by targeting the downstream effector of FKBP7.

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