Leishmania est un parasite protozoaire eucaryote unicellulaire qui infecte plus de 1.6 millions de personnes chaque année dans plus de 98 pays. Aucun vaccin humain est actuellement disponible et peu de traitements efficaces sont utillisés pour lutter contre le large spectre de pathologies causées par Leishmania. Récemment, l’étude du contrôle de la qualité des protéines chez Leishmania infantum a révélé que DDX3, une DEAD-box hélicase à ARN dotée de multiples fonctions dans le métabolisme de l’ARN et la signalisation cellulaire, joue un rôle central dans le contrôle de qualité des protéines dans la mitochondrie. Une étude plus approfondie de ce mécanisme a révélé des interactions potentielles de DDX3 avec des composantes clés de la réponse cellulaire au stress, en particulier avec une protéine de la famille des AAA + ATPases, VCP/p97/Cdc48. Comme VCP/p97/Cdc48 participe à de multiples étapes dans le contrôle de qualité des protéines en utilisant son hydrolyse de l’ATP pour séparer les protéines ubiquitinées de leurs partenaires et les acheminer au protéasome 26S pour dégradation, nous avons émis l’hypothèse que l’homologue très conservé chez Leishmania, LiVCP, pourrait agir de la même façon. Cette étude a permis la caractérisation fonctionnelle de l’homologue VCP chez Leishmania, son rôle dans la réponse du parasite au stress et sa survie dans les macrophages, ses interactions potentielles avec d’autres partenaires dont des cofacteurs clés, ainsi que la modélisation 3D des interactions LiVCP-cofacteurs. En utilisant des mutants génétiquement générés ayant moins de copies du gène LiVCP ou des mutants dominants négatifs avec une activité VCP altérée, nous avons démontré que LiVCP est un gène essentiel et que les mutants VCP sont incapables de survivre sous le shock de la chaleur et présentent un déficit de croissance très marqué chez les amastigotes. De plus, nous avons montré une forte accumulation de protéines polyubiquitinées et une sensibilité accrue au stress protéotoxique chez ces mutants, soutenant la fonction de chaperone sélective de l'ubiquitine de LiVCP. Grâce à des analyses in silico et à la «protéomique en réseau» en utilisant des études de co-immunoprécipitation et de spectrométrie de masse (LC-MS / MS), nous avons établi le premier réseau protéique de VCP chez les parasites protozoaires et déterminé que p47, FAF2, UFD1, PUB1 et l’hétérodimère NPL4-UFD1 étaient les principaux cofacteurs de LiVCP. Enfin, nos travaux nous ont permis de faire progresser nos connaissances générales sur la protéine essentielle VCP et le contrôle de la qualité des protéines chez Leishmania et d’indiquer quelques perspectives intéressantes pour approfondir notre compréhension sur ces mécanismes importants non seulement chez Leishmania mais aussi chez d’autres trypanosomatides. / Leishmania is a unicellular eukaryotic protozoan parasite that infects over 1.6 million people each year in more than 98 countries. No human vaccine is currently available and few effective treatments are used to combat the broad spectrum of diseases caused by Leishmania. Recently, studies on Protein Quality Control in Leishmania infantum revealed that the multifunctional DEAD-box RNA helicase DDX3 involved among others in RNA metabolism and cell signaling plays a central role in mitochondrial protein quality control. Further studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly with the conserved AAA+ ATPase VCP/ p97/Cdc48. As VCP is associated with many ubiquitin-dependent cellular pathways that are central to protein quality control in other eukaryotic systems using its ATP hydrolysis to separate ubiquitinated proteins from their partners and bring them to the 26S proteasome for degradation, we hypothesized that the Leishmania highly conserved counterpart, LiVCP, might act in similar way. This study enabled the functional characterization of the Leishmania VCP homolog, its role in the parasite's response to stress and survival inside macrophages, its potential interactions with other partners including key VCP cofactors, and the homology 3D modeling of LiVCP-cofactor interactions. Using genetically engineered mutants with fewer copies of the LiVCP gene or dominant negative mutants with altered VCP activity, we demonstrated that LiVCP is an essential gene and that VCP mutants are unable to survive under heat stress and exhibit a very marked growth defect in amastigotes. In addition, we showed a high accumulation of polyubiquitinated proteins and increased susceptibility to proteotoxic stress in these mutants, supporting that LiVCP has an ubiquitin selective chaperone function. Using "network proteomics" analyses by co-immunoprecipitation and mass spectrometry (LC-MS/MS) studies, we established the first VCP protein network in protozoan parasites and determined p47, FAF2, UFD1, PUB1 and the NPL4-UFD1 heterodimer as the major cofactors of LiVCP. Overall, our work allowed us to advance general knowledge of the essential role of VCP in Leishmania protein quality control and to propose some interesting perspectives to deepen our understanding of these important pathways not only in Leishmania but also in other trypanosomatids.

P1B-type ATPases transport a number of monovalent and divalent heavy metals (Cu+, Cu2+, Ag+, Zn2+, Cd2+, Pb2+ and Co+2) across biological membranes. These ATPases are found in archea, bacteria and eukaryotes and are one of the key elements required for maintaining metal homeostasis. Plants have an unusually high number of P1B-type ATPases with distinct metal selectivity compared to other eukaryotes that usually have one or two Cu+-ATPases. Higher plants are the only eukaryotes where Zn2+-ATPases have been identified. Towards understanding the physiological roles of plant Zn2+-ATPases, we characterized Arabidopsis thaliana HMA2. We expressed HMA2 in yeast and measured the metal dependent ATPase activity in membranes. We showed that HMA2 is a Zn2+-ATPase that is also activated by Cd2+. Zn2+ transport determinations showed that this enzyme drives the efflux of metal from the cytoplasm. Analysis of HMA2 mRNA levels showed that the enzyme is present in all plant organs. We analyzed the effect of removal of HMA2 full-length transcript in whole plants by gene knock out. Although hma2 mutants did not show a different visible phenotype from the wild type plants, we observed increased levels of Zn2+ or Cd2+. The observed phenotype of hma2 mutants and plasma membrane location of HMA2, mainly in vasculature (Hussain et al., 2004), indicates that this ATPase might have a central role in Zn2+ uploading into the phloem. P1B-type ATPases have cytoplasmic regulatory metal binding domains (MBDs) in addition to transmembrane metal binding sites (TMBDs). Plant Zn2+-ATPases have distinct sequences in both their N- and C-termini that might contribute to novel metal binding sites. These ATPases contain long C-terminal sequences rich in CC dipeptides and His repeats. Removal of the C-terminus (C-MBD) of HMA2 leads to a 50% reduction in the enzyme turnover suggesting a regulatory role for this domain. Atomic Absorption Spectroscopy (AAS) analysis showed that Zn2+ binds to C-MBD with a stoichiometry of three (3 Zn/C-MBD). Chemical modification studies and Zn K-edge X-ray Absorption Spectroscopy (XAS) of Zn-C-MBD showed that Zn2+ is likely coordinated by His in two sites and the third site slightly differs from the others involving a Cys together with three His. All plant Zn2+-ATPases lack the typical CXXC signature sequences observed in Cu+-ATPases and some bacterial Zn2+-ATPases N-terminus metal binding domains (N-MBDs). Instead, these have conserved CCXXE sequences. Truncation of HMA2 N-MBD results in a 50% decrease in enzyme Vmax suggesting that N-MBD is also a regulatory domain. The results indicate that the N-MBD binds Zn2+ with a stoichiometry of one (1 Zn/N-MBD). Metal binding analysis of individual N-MBD mutants Cys17Ala, Cys18Ala and Glu21Ala/Cys prevented Zn+2 binding to HMA2 N-MBD suggesting the involvement of all these residues in metal coordination. ATPase activity measurements with HMA2 carrying the mutations Cys17Ala, Cys18Ala and Glu21Ala/Cys showed a reduction in the enzyme activity similar to that observed the truncated protein indicating that the enzyme activity reduction observed in the N-terminus truncated forms of the enzyme is related to the removal of the metal binding capability. Summaryzing, these studies show the central role of HMA2 in plant Zn2+ homeostasis. They also describe the mechanism and direction of Zn2+ transport. Finally, they establish the presence of novel metal binding domains in the cytoplasmic portion of the enzyme. Metal binding to these domains is required for full enzymatic activity.

Qi Wei. / "November 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 158-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Xue, Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 95-102). / Abstract also in Chinese. / Thesis committe --- p.2 / Statement --- p.3 / Abstract --- p.4 / Acknowledgement --- p.8 / General abbreviations --- p.10 / Abbreviations of chemicals --- p.13 / List of figures --- p.15 / List of tables --- p.16 / Table of contents --- p.17 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Impact of bacterial blight on rice production --- p.25 / Chapter 1.2 --- The plant immune system --- p.25 / Chapter 1.2.1 --- Preformed resistance --- p.25 / Chapter 1.2.2 --- PAMP triggered immunity (PTI) --- p.26 / Chapter 1.2.3 --- Effecter triggered immunity (ETI) --- p.27 / Chapter 1.2.3.1 --- R genes --- p.27 / Chapter 1.2.3.2 --- Hypersensitive responses (HR) --- p.27 / Chapter 1.2.3.3 --- Systemic acquired resistance (SAR) --- p.28 / Chapter 1.2.3.3.1 --- Salicylic acid is required for SAR establishment --- p.28 / Chapter 1.2.3.3.2 --- Involvement of lipid-based molecules in SAR signaling --- p.28 / Chapter 1.2.3.3.3 --- NPR1: the master regulator of SAR --- p.29 / Chapter 1.2.3.3.4 --- Expression of pathogenesis related (PR) genes --- p.29 / Chapter 1.2.4 --- Interaction between SA and JA --- p.29 / Chapter 1.2.5 --- Other important signaling components in plant defense responses --- p.30 / Chapter 1.2.5.1 --- G proteins --- p.30 / Chapter 1.2.5.2 --- G proteins in defense responses --- p.30 / Chapter 1.3 --- OsGAPl is a C2 (protein kinase C conserved region 2) domain harboring GTPase activating protein --- p.32 / Chapter 1.4 --- OsYchFl is a GTPase and an interacting partner of OsGAPl --- p.32 / Chapter 1.5 --- Hypothesis and objectives of this research --- p.33 / Chapter Chapter 2 --- materials and methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Chemicals and reagents --- p.39 / Chapter 2.1.2 --- Commercial kits --- p.40 / Chapter 2.1.3 --- Primers used --- p.41 / Chapter 2.1.4 --- Equipment and facilities used: --- p.47 / Chapter 2.1.5 --- "Buffer, solution, gel and medium:" --- p.47 / Chapter 2.2 --- Methods: --- p.51 / Chapter 2.2.1 --- Culture of bacterial strains --- p.51 / Chapter 2.2.2 --- Composition of medium used in this work for cultivating bacterial strains: --- p.51 / Chapter 2.2.3 --- Plant growth and treatment --- p.52 / Chapter 2.2.3.1 --- Surface sterilization of Arabidopsis thaliana seeds --- p.52 / Chapter 2.2.3.2 --- Seed germination and Arabidopsis plant growth --- p.52 / Chapter 2.2.4 --- Generation of transgenic Arabidopsis --- p.53 / Chapter 2.2.4.1 --- Agrobacterium-mediated Arabidopsis transformation --- p.53 / Chapter 2.2.5 --- Pathogen inoculation test --- p.54 / Chapter 2.2.6 --- Molecular cloning --- p.54 / Chapter 2.2.6.1 --- DNA sequencing: --- p.55 / Chapter 2.2.6.2 --- Transformation of E. coli strains: --- p.55 / Chapter 2.2.6.3 --- Transformation of Agrobacteria by electroporation --- p.55 / Chapter 2.2.7 --- DNA and RNA extraction --- p.56 / Chapter 2.2.7.1 --- Plasmid DNA extraction from bacterial cells --- p.56 / Chapter 2.2.7.2 --- Genomic DNA extraction from plant tissues --- p.56 / Chapter 2.2.7.3 --- RNA extraction from plant tissues --- p.56 / Chapter 2.2.8 --- Northern blot --- p.57 / Chapter 2.2.9 --- Subcellular localization studies --- p.58 / Chapter 2.2.9.1 --- Transformation of tobacco BY-2 cells --- p.58 / Chapter 2.2.9.2 --- Maintenance of transgenic tobacco BY-2 cells --- p.59 / Chapter 2.2.9.3 --- Confocal microscopy --- p.59 / Chapter 2.2.9.4 --- Electron microscopy --- p.59 / Chapter 2.2.10 --- Bimolecular fluorescence complementation studies (BiFC) --- p.60 / Chapter 2.2.10.1 --- Construct making --- p.61 / Chapter 2.2.10.2 --- Preparation of rice protoplasts --- p.61 / Chapter 2.2.10.3 --- PEG-mediated transfection --- p.62 / Chapter 2.2.10.4 --- Detection of protein-protein interaction --- p.62 / Chapter Chapter 3 --- Results / Chapter 3.1 --- OsGAPl interacts with OsYchFl in vivo --- p.63 / Chapter 3.1.1 --- Construction of vectors for BiFC transient assay in rice protoplasts --- p.64 / Chapter 3.1.2 --- BiFC assay in rice protoplasts revealed in vivo interaction between the OsGAPl and the OsYchFl proteins --- p.66 / Chapter 3.2.1 --- Subcellular localization of OsGAPl --- p.68 / Chapter 3.2.2 --- Localization of OsGAPl and OsYchFl in rice leaves revealed by electron microscopy --- p.70 / Chapter 3.3 --- Functional characterization of OsYchFl / Chapter 3.3.1 --- Characterization of Arabidopsis YchF1 knockdown mutant --- p.75 / Chapter 3.3.2 --- Complementation of AtYchF1 knockdown Arabidopsis --- p.77 / Chapter 3.3.3.1 --- Pathogen inoculation test --- p.80 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Significance of the project --- p.85 / Chapter 4.2 --- In vivo interaction between OsGAPl and OsYchFl --- p.86 / Chapter 4.3 --- OsGAPl is located either inside the cytosol or on the plasma membrane in transgenic tobacco BY-2 cells --- p.87 / Chapter 4.4 --- Study of wounding effect on the subcellular localization of OsGAPl and OsYchFl at whole plant level by EM --- p.88 / Chapter 4.5 --- OsYchFl functions as a negative regulator of defense responses in A.thaliana --- p.90 / Chapter 4.6 --- Conclusion --- p.92 / References --- p.95 / Appendix --- p.103

No description available.

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu. / x, 85 leaves : ill. (some col.) ; 29 cm

Two enzymatic systems from the lactic acid bacterium Oenococcus oeni, isolated from wine, have been studied. The first one is the H+-ATPase for which the activity was characterized under various conditions of growth. The activity gradually increased by l.6 to 1.9-fold upon inoculation at pH 3.5. The H+-ATPase activity did not vary significantly in function of the growth rate or with and without malic acid. However, acidification of the medium in the absence of malic acid induced the activity by 1.5 to 2.2-fold depending on the initial pH. The partially cloned H+-ATPase genes shared high homologies with those from other bacterial F0F1-ATPases. A mRNA of about 7 kb was detected by Northern blot and its size suggests that the genetic organization of O. oeni atp operon is similar to most F0F 1-ATPases. Furthermore, the amount of atp mRNA was shown to increase in acidic conditions. O. oeni H +-ATPase activity was pH-inducible and regulation of the expression seems to occur at the level of mRNA synthesis. Thus, the results confirmed the proposed role of the H+-ATPase in acid tolerance in O. oeni. / The second system studied was a chromosome-encoded P-type ATPase (CopB) and its putative transcriptional regulator (CopR). The copB gene encodes a protein showing great similarities with other Cu2+-ATPases of the CPx-type family of heavy-metal ATPases like Enterococcus hirae copB. Another gene (copR) was found 250 bp upstream of copB and displays great similarities with proteins of the MecI/BlaI family of transcriptional regulators, including En. hirae CopY repressor. O. oeni was shown to be highly resistant to copper and growth occurred in up to 30 mM CuSO4. Northern blot analyses indicated that the amount of copB mRNA increased upon a 0.2 to 4.0 mM copper stress suggesting that expression of the enzyme might be regulated at the level of mRNA synthesis. Whether CopR is involved in this regulation remains to be determined, but the results suggest that copRB genes might be involved in copper resistance in O. oeni.

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed on Aug. 20, 2009) Includes bibliographical references. Also issued in print.