Yes / The fate of secretory and membrane proteins that mislocalize to the cytosol is decided by a collaboration between cochaperone SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the BAG6 complex, whose operation relies on multiple transient and subtly discriminated interactions with diverse binding partners. These include chaperones, membrane-targeting proteins and ubiquitination enzymes. Recently a direct interaction was discovered between SGTA and the proteasome, mediated by the intrinsic proteasomal ubiquitin receptor Rpn13. Here, we structurally and biophysically characterize this binding and identify a region of the Rpn13 C-terminal domain that is necessary and sufficient to facilitate it. We show that the contact occurs through a carboxylate clamp-mediated molecular recognition event with the TPR domain of SGTA, and provide evidence that the interaction can mediate the association of Rpn13 and SGTA in a cellular context. / RLI was supported by MRC New Investigator Research Grant: G0900936. RLI and SH are funded by BBSRC grants: BB/L006952/1 and BB/L006510/1 respectively. RLI is funded by BBSRC grant: BB/N006267/1. AT is funded by BBSRC grant: BB/J014567/1. ILT was the recipient of a Wellcome Trust Vacation Scholarship 2015. NMR experiments were performed at the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trust

Yes / Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects. However, the DUBs that play a role in this process have not been identified. In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5. We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.

Les mécanismes moléculaires contrôlant les rythmes circadiens sont conservés parmi les organismes des différents règnes (plantes, animaux et champignons). Ils se composent de boucles de rétroaction où un complexe d’activation transcriptionnelle, l’hétérodimère CLK/CYC chez la drosophile, entraîne l'expression des répresseurs de son activité, les gènes et protéines PER et TIM chez la mouche. De manière importante, la période de l'oscillateur dépend en grande partie par des mécanismes post-transcriptionnels qui régulent l’accumulation et l'activité des composantes positifs et négatifs de la boucle. Bien que de nombreux partenaires d'interaction modifiant les composants d'horloge de base ont déjà pu être isolés, le schéma reste encore incomplet. Dans le cadre de la recherche de nouveaux composants de cette horloge, nous avons réalisé un crible comportemental basé sur l'expression ciblée de transgènes ARNi dirigés contre la moitié du génome de Drosophila melanogaster. Cinquante-quatre nouveaux gènes putatifs ont pu être identifiés. Au cours de ce travail, j'ai étudié le rôle de deux d’entre eux, sélectionnés pour les forts défauts comportementaux de l'expression de leur transgène ARNi. Le gène CG12082 de la drosophile est l’orthologue de l’Ubiquitin-specific protéase 5 (USP5) chez l’homme. La dérégulation d’Usp5 retarde les oscillations de la protéine PER dans les neurones d'horloge et allonge la période d'activité locomotrice des mouches. Chez les mouches ARNi Usp5, des formes à haut poids moléculaire des protéines PER et TIM s'accumulent pendant le matin, alors qu’elles sont normalement dégradées chez les contrôles. On a pu montrer que Usp5 participe directement à la dégradation de la protéine PER, indépendamment de TIM. En accord avec le rôle décrit pour l’orthologue humaine, Usp5 serait susceptible de contrôler la dégradation des protéines par son activité de démontage des chaînes libres de polyubiquitine présents dans la cellule, qui peuvent entrer en compétition avec les protéines ubiquitinylées pour la reconnaissance au niveau du protéasome, bloquant leur dégradation. La majorité des travaux ont porté sur un gène isolé au cours de notre crible, Strip, dont les fonctions étaient encore inconnues. Strip interagit avec Cka, une nouvelle sous-unité régulatrice de l’enzyme phosphatase PP2A. La dérégulation à la fois de Strip et/ou de Cka amène à des phénotypes comportementaux de période longue. D’un point de vue moléculaire, des formes hyper-phosphorylées de la protéine CLK s’accumulent dans la matinée quand Cka et/ou Strip sont perturbées. La dérégulation des activités générales de PP2A produit également une hyper-phosphorylation de CLK le matin, indiquant que, grâce à Cka/Strip, les complexes PP2A contrôlent la déphosphorylation de CLK à la fin du cycle. Il est connu que les formes hyper-phosphorylés de CLK sont transcriptionnellement inactives. En effet, la transcription des gènes tim et vrille, cibles de CLK, est fortement réduite dans les mouches ARNi Cka. En plus de PP2A/Cka, des complexes PP2A contenant une autre sous-unité régulatrice, Wdb, ont été montré pour déstabiliser CLK en culture des cellules (Kim et Edery, 2006). Nous montrons que la dérégulation de Wdb affecte la stabilité du CLK également dans la mouche adulte, sans toutefois induire aucun effet apparent sur sa phosphorylation. En conclusion, deux complexes PP2A différents agissent sur la protéine CLK : le complexe PP2A/Cka/Strip contrôle la déphosphorylation de CLK et sa réactivation, tandis que PP2A/Wdb affecte la stabilité de CLK indépendamment ou après PP2A/Cka. Ces résultats enrichissent l’étude de la régulation post-traductionnelle de la protéine CLK, qui était largement mal connue.Pour conclure, cette étude a permis de décrire deux nouveaux composants de la boucle moléculaire qui contrôle les rythmes circadiens chez la mouche du vinaigre, Drosophila melanogaster. / The molecular mechanism underlying circadian rhythms is conserved among organisms and consists of feedback loops where a transcriptional activating complex (the CLOCK (CLK)/CYCLE (CYC) heterodimer in Drosophila) drives the expression of the repressors of its activity (the period (per) and timeless (tim) genes and proteins in Drosophila). Importantly, the pace of the oscillator largely depends on post-transcriptional mechanisms that regulate the accumulation and activity of both the positive and negative components of the loop. A number of interacting partners that modify core clock components have already been isolated, but more are expected. Looking for new clock components, we set up a behavioral screen based on targeted expression of RNAi transgenes directed to half of the Drosophila genome. 54 putative new clock genes have been identified. Among them, some were independently reported to function within the fruit fly molecular clock, thus validating the screen. In this work, I investigated the circadian role of additional “positive” genes, selected for the strong behavioral defect induced by the expression of the corresponding RNAi. The CG12082 gene codes for the fruit fly ortholog of the human Ubiquitin-specific protease 5 (USP5). Downregulation of USP5 in clock cells lengthens the period of locomotor activity of flies as well as PER protein oscillations in clock neurons. High molecular weight forms of PER and TIM proteins accumulate during the morning after USP5 knockdown, while these forms are degraded in controls. In addition, TIM is not stabilized in the absence of PER, while PER still accumulate in the absence of TIM. Therefore, USP5 directly participates in the degradation of the PER protein and, later, of the TIM protein at the end of the cycle. Being a deubiquitinylase enzyme, USP5 may directly deubiquitinate PER. However, accordingly to the role described for the human ortholog, USP5 likely controls protein degradation through the disassembling of the unanchored polyubiquitin chains present in the cell that could compete with ubiquitinated-PER for proteasome recognition and subsequent breakdown.The majority of the work has focused on an unknown gene isolated in the screen, that, accordingly to the human homolog, we named STRIP. We show that STRIP interacts with Connector of Kinase to AP-1 (CKA), a novel regulatory subunit for the PP2A phosphatase holoenzyme, both in insect S2 cells and in fly head extracts. Downregulation of both STRIP and/or CKA causes long-period behavioral phenotypes and high molecular weight forms of the CLK protein to accumulate in the morning. Perturbation of general PP2A activities also produces hyper-phosphorylated CLK in the morning indicating that, through CKA/STRIP, PP2A complexes controls CLK dephosphorylation at the end of the cycle. Hyper-phosphorylated CLK forms are transcriptionally inactive. Accordingly, transcription of the tim and vrille (vri) CLK targets is strongly reduced in Cka-RNAi fly head extracts. PP2A complexes containing the Widerborst (WDB) regulatory subunits were already shown to affect CLK stability in insect S2 cells (Kim and Edery, 2006). We show that WDB downregulation also affects the stability of CLK in fly head extracts, but has no apparent effects on CLK phosphorylation. Therefore, we could describe two different PP2A complexes acting on the CLK protein: PP2A/CKA/STRIP complex controls CLK dephosphorylation and reactivation, while PP2A/WDB affects CLK stability independently or after PP2A/CKA functions. Moreover, STRIP, but not CKA, downregulation affects the stability of PER, indicating that STRIP possesses some functions unrelated to CKA. In conclusion, this work has allowed the isolation of new components of the Drosophila molecular clock. In particular, we give evidence for a double role for the PP2A phosphatase in modulating the activity and stability of the CLK protein, the regulation of which is not well understood yet.

Chan, Ho Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 263-283). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Abbreviations --- p.xvii / List of Figures --- p.xx / List of Tables --- p.xxv / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- SUMO (Small Ubiquitin-like Modifier) and SUMOylation --- p.1 / Chapter 1.1.1 --- "Ubiquitin, Ubiquitin-like proteins and SUMO isoforms" --- p.2 / Chapter 1.1.2 --- SUMO cycle --- p.5 / Chapter 1.1.2.1 --- SUMO conjugation consensus sequence --- p.5 / Chapter 1.1.2.2 --- SUMO maturation --- p.6 / Chapter 1.1.2.3 --- SUMO conjugation cascade --- p.7 / Chapter 1.1.2.4 --- SUMO deconjugation --- p.9 / Chapter 1.1.3 --- Mode of SUMO action --- p.12 / Chapter 1.1.4 --- Biological functions of SUMO --- p.13 / Chapter 1.1.4.1 --- SUMO in cancer --- p.14 / Chapter 1.2 --- Human cervical cancer and human papillomavirus (HPV) --- p.17 / Chapter 1.2.1 --- Infectious cycle of HPV-16 --- p.18 / Chapter 1.2.1.1 --- Viral entry --- p.18 / Chapter 1.2.1.2 --- Maintenance --- p.18 / Chapter 1.2.1.3 --- Deregulation of cell cycle --- p.19 / Chapter 1.2.1.4 --- Amplification and virion release --- p.20 / Chapter 1.2.2 --- Viral cancer induction --- p.22 / Chapter 1.2.2.1 --- Integration into the host genome --- p.22 / Chapter 1.2.2.2 --- Viral oncoproteins E6 and E7 --- p.23 / Chapter 1.2.3 --- SUMOylation and HPV --- p.24 / Chapter 1.2.3.1 --- Known examples of virus-host SUMOylation system interaction --- p.24 / Chapter 1.2.3.2 --- Other possible mode of virus-SUMO interaction --- p.26 / Chapter 1.3 --- A novel labeling method: biarsenical-tetracysteine labeling in SUMO study --- p.28 / Chapter 1.3.1 --- Potential use of 2As-4Cys system in SUMO studies --- p.31 / Chapter 1.3.2 --- Potential use of 2As-4Cys system in SUMO proteomics --- p.31 / Chapter 1.4 --- Objectives of the present study --- p.34 / Chapter Chapter II --- Proteomics investigation of SUMOylation in human cervical carcinoma cell line HeLa --- p.35 / INTRODUCTION --- p.35 / Chapter 2.1 --- MATERIALS --- p.37 / Chapter 2.1.1 --- Vectors for expression of SUMO and SUMOylation enzymes in E. coli --- p.37 / Chapter 2.1.2 --- E.coli cell strains --- p.38 / Chapter 2.1.3 --- Mammalian cell lines --- p.39 / Chapter 2.1.4 --- E.coli growth mediums --- p.40 / Chapter 2.1.5 --- Mammalian cell growth medium --- p.41 / Chapter 2.1.6 --- Reagents and buffers --- p.41 / Chapter 2.1.6.1 --- Reagents and buffers for molecular cloning --- p.41 / Chapter 2.1.6.2 --- Reagents and buffers for E.coli protein expression --- p.43 / Chapter 2.1.6.3 --- Reagents and buffers for mammalian cell culture --- p.44 / Chapter 2.1.6.4 --- Reagents and buffers for Western blot study --- p.45 / Chapter 2.1.7 --- Reagents and solutions for two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) sample preparation --- p.46 / Chapter 2.1.7.1 --- Reagents and solutions for 2-DE --- p.46 / Chapter i. --- 2-DE sample preparation --- p.46 / Chapter ii. --- First dimensional gel electrophoresis -isoelectric focusing (IEF) --- p.46 / Chapter iii. --- Second dimensional gel electrophoresis -SDS-PAGE --- p.47 / Chapter iv. --- Silver staining --- p.47 / Chapter 2.1.7.2 --- Reagents and solutions for mass spectrometry sample preparation --- p.48 / Chapter i. --- Destaining of silver stained gel spots --- p.48 / Chapter ii. --- Trypsin digestion --- p.48 / Chapter iii. --- Peptide extraction --- p.48 / Chapter iv. --- Desalting and concentration of peptide mixture --- p.49 / Chapter 2.2 --- METHODS --- p.50 / Chapter 2.2.1 --- Molecular cloning of SUMO-1 into pET-28m and pHM6 vectors --- p.50 / Chapter 2.2.1.1 --- Design of primers for the cloning of SUMO-1 --- p.50 / Chapter 2.2.1.2 --- DNA amplification by polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.1.3 --- DNA extraction from agarose gels --- p.52 / Chapter 2.2.1.4 --- Restriction digestion of vectors and purified PCR products --- p.54 / Chapter 2.2.1.5 --- Ligation of SUMO cDNA into expression vector pET-28m and pHM6 --- p.55 / Chapter 2.2.1.6 --- Preparation of competent cells --- p.56 / Chapter 2.2.1.7 --- Transformation of ligated mixture into competent DH5a --- p.56 / Chapter 2.2.1.8 --- Preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.1 --- Mini-preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.2 --- Midi-preparation of plasmid DNA --- p.58 / Chapter 2.2.1.8.3 --- DNA quantification and quality measurement --- p.60 / Chapter 2.2.2 --- "Expression of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1 with E.coli" --- p.60 / Chapter 2.2.3 --- "Purification of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1" --- p.62 / Chapter 2.2.3.1 --- Affinity chromatography --- p.65 / Chapter 2.2.3.1.1 --- Ni-NTA affinity chromatography --- p.65 / Chapter 2.2.3.1.2 --- Heparin affinity chromatography --- p.66 / Chapter 2.2.3.1.3 --- Glutathione affinity chromatography --- p.66 / Chapter 2.2.3.1.4 --- Amylose affinity chromatography --- p.67 / Chapter 2.2.3.2 --- Ion exchange chromatography --- p.68 / Chapter 2.2.3.2.1 --- Anion exchange chromatography --- p.68 / Chapter 2.2.3.2.2 --- Cation exchange chromatography --- p.68 / Chapter 2.2.3.3 --- Size exclusion chromatography --- p.69 / Chapter 2.2.3.4 --- Purification strategies --- p.70 / Chapter 2.2.3.4.1 --- Purification of His6-tagged SUMO --- p.70 / Chapter 2.2.3.4.2 --- Purification of His6-tagged TDG --- p.71 / Chapter 2.2.3.4.3 --- Purification of His6-tagged ubc9 --- p.72 / Chapter 2.2.3.4.4 --- Purification of GST-tagged El --- p.73 / Chapter 2.2.3.4.5 --- Purification of MBP-tagged Prdx 1 --- p.74 / Chapter 2.2.4 --- HeLa and C-33A cell culturing and protein extraction --- p.75 / Chapter 2.2.4.1 --- HeLa and C-33A cell culturing --- p.75 / Chapter 2.2.4.2 --- Protein extraction for in vitro SUMOylation assay --- p.76 / Chapter 2.2.5 --- Protein quantification with Bradford assay --- p.76 / Chapter 2.2.6 --- In vitro SUMO conjugation assay --- p.77 / Chapter 2.2.6.1 --- In vitro SUMO conjugation system optimization --- p.77 / Chapter 2.2.6.2 --- In vitro SUMO conjugation of HeLa cell extract --- p.78 / Chapter 2.2.7 --- Transient transfection of pHM6-SUMO-l into HeLa cells and protein extraction from HeLa cells --- p.79 / Chapter 2.2.7.1 --- Transfection with lipofection method --- p.79 / Chapter 2.2.7.2 --- Determination of transfection efficiency --- p.80 / Chapter 2.2.7.3 --- Whole cell protein extraction of transfected cells --- p.81 / Chapter 2.2.8 --- Protein quantification with BCA assay --- p.81 / Chapter 2.2.9 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.83 / Chapter 2.2.10 --- Western blot analysis --- p.84 / Chapter 2.2.10.1 --- Electro-transfer blotting --- p.84 / Chapter 2.2.10.2 --- Immunoblotting with antibodies --- p.84 / Chapter 2.2.10.3 --- ECL detection --- p.85 / Chapter 2.2.10.4 --- Mild stripping for re-probing --- p.86 / Chapter 2.2.11 --- Two-dimensional gel electrophoresis (2-DE) --- p.86 / Chapter 2.2.11.1 --- Sample preparation --- p.86 / Chapter 2.2.11.2 --- First dimension gel electrophoresis -isoelectric focusing (IEF) --- p.87 / Chapter 2.2.11.3 --- Second dimension gel electrophoresis -SDS-PAGE --- p.88 / Chapter 2.2.11.3.1 --- Strip equilibration --- p.88 / Chapter 2.2.11.3.2 --- 16 x 18cm SDS-PAGE --- p.88 / Chapter 2.2.11.4 --- Visualization of proteins on SDS-polyacrylamide gel --- p.90 / Chapter 2.2.11.4.1 --- Silver staining --- p.90 / Chapter 2.2.11.4.2 --- Coomassie Blue® R250 staining --- p.91 / Chapter 2.2.12 --- Sample preparation for mass spectrometry analysis --- p.92 / Chapter 2.2.12.1 --- Destaining and trypsin digestion --- p.92 / Chapter 2.2.12.2 --- Extraction of peptide mixture --- p.93 / Chapter 2.2.12.3 --- Desalting and concentration of peptide mixture --- p.93 / Chapter 2.3 --- RESULTS --- p.95 / Chapter 2.3.1 --- Construction of recombinant pET-28m-SUMO-l and pHM6-SUMO-l --- p.95 / Chapter 2.3.2 --- "Purification of His6-tagged SUMO, ubc9, TDG and GST-tagged El" --- p.98 / Chapter 2.3.2.1 --- Purification of His6-SUMO --- p.98 / Chapter 2.3.2.2 --- Purification of His6-TDG --- p.101 / Chapter 2.3.2.3 --- Purification of His6-ubc9 --- p.104 / Chapter 2.3.2.4 --- Purification of GST-El --- p.106 / Chapter 2.3.3 --- In vitro SUMO conjugation assay --- p.108 / Chapter 2.3.3.1 --- Optimization of in vitro SUMO conjugation system --- p.108 / Chapter 2.3.3.2 --- In vitro SUMO conjugation of HeLa cell protein extract --- p.111 / Chapter 2.3.3.2.1 --- Protein extraction for in vitro sumoylation assay --- p.111 / Chapter 2.3.3.2.2 --- In vitro SUMOylation of HeLa cell lysate --- p.114 / Chapter 2.3.4 --- Differential proteomes of control and in vitro SUMOylated HeLa total cellular extract --- p.116 / Chapter 2.3.4.1 --- Mass spectrometric identification of differential protein candidates --- p.123 / Chapter 2.3.5 --- Overexpression of SUMO-1 in HeLa cells by transient transfection --- p.127 / Chapter 2.3.6 --- Differential proteomes of total cellular protein extract from control and SUMO-1 transfected HeLa cells --- p.128 / Chapter 2.3.6.1 --- Mass spectrometric identification of differential protein candidates --- p.132 / Chapter 2.4 --- Proteins identified in proteomic study with in vitro SUMOylation -Analysis of protein candidate --- p.133 / Chapter 2.4.1 --- Proteins identified from the in vitro investigation --- p.133 / Chapter 2.4.2 --- Verification of putative SUMO substrate Prdx 1 --- p.139 / Chapter 2.4.2.1 --- Purification of Prdx 1 --- p.139 / Chapter 2.4.2.2 --- In vitro SUMOylation of Prdx 1 --- p.142 / Chapter 2.4.3 --- Highlights of the proteins identified --- p.145 / Chapter 2.4.3.1 --- DJ-1 protein --- p.145 / Chapter 2.4.3.2 --- nm23A --- p.145 / Chapter 2.4.3.3 --- v-crk protein of CT10 --- p.146 / Chapter 2.4.3.4 --- Annexin I --- p.146 / Chapter 2.4.3.5 --- "Enolase 1, aldolase A, triosephosphate isomerase (TIM) and phosphoglycerate mutase 1" --- p.147 / Chapter 2.4.3.6 --- CyclophilinA(CypA) --- p.148 / Chapter 2.4.3.7 --- Stress induced phosphoprotein 1 (Stip 1) --- p.148 / Chapter 2.4.3.8 --- TSA and peroxiredoxin 1 (Prdx 1) --- p.149 / Chapter 2.5 --- Proteins identified in proteomic study with overexpression of SUMO-1 in HeLa cells -Analysis of protein candidate --- p.150 / Chapter 2.5.1 --- Proteins identified from the in vivo investigation --- p.150 / Chapter 2.5.2 --- Verification of upregulation of keratin 17 --- p.157 / Chapter 2.5.2.1 --- Immunoblotting against keratin 17 --- p.157 / Chapter 2.5.3 --- Highlights of the proteins identified --- p.159 / Chapter 2.5.3.1 --- "Heat shock proteins (Hsp 60, 70 and 27)" --- p.159 / Chapter 2.5.3.2 --- 14-3-3σ protein (SFN protein) --- p.161 / Chapter 2.5.3.3 --- PDZ-RGS3 --- p.162 / Chapter 2.5.3.4 --- "Keratins 8, 17" --- p.163 / Chapter 2.5.3.5 --- XIAP-1 --- p.164 / Chapter 2.5.3.6 --- ISG15 --- p.164 / Chapter 2.6 --- DISCUSSION --- p.166 / Chapter Chapter III --- Characterization of a novel fluorescent labeling method: Biarsencial-tetracysteine labeling in SUMO study --- p.182 / INTRODUCTION --- p.182 / Chapter 3.1 --- MATERIALS --- p.184 / Chapter 3.1.1 --- "Molecular cloning, protein expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1" --- p.184 / Chapter 3.1.2 --- Mammalian cell culture and transient transfection of pHM6-4Cysl-SUMO-1 and pHM6-4Cys2-SUMO-l into HeLa cells --- p.184 / Chapter 3.1.3 --- Reagents and buffers --- p.184 / Chapter 3.1.3.1 --- Reagents and buffers for Lumio´ёØ in-gel labeling --- p.184 / Chapter 3.1.3.2 --- Reagents and buffers for Lumio´ёØ in cell labeling --- p.185 / Chapter 3.1.3.3 --- Reagents and buffers for immunostaining --- p.186 / Chapter 3.2 --- METHODS --- p.187 / Chapter 3.2.1 --- Molecular cloning of tetracysteine-tagged SUMO (4Cys-SUMO) into pET-28m and pHM6 vectors --- p.187 / Chapter 3.2.1.1 --- Design of primers and oligonucleotides encoding tetracysteine tag --- p.187 / Chapter 3.2.1.1.1 --- For 4Cysl-SUMO-1 --- p.187 / Chapter 3.2.1.1.2 --- For 4Cys2-SUMO-l --- p.188 / Chapter 3.2.1.2 --- DNA amplification of 4Cysl-SUMO-1 by Polymerase chain reaction (PCR) --- p.189 / Chapter 3.2.1.3 --- Restriction digestion of vectors and purified PCR products of 4Cysl-SUMO-1 --- p.191 / Chapter 3.2.1.4 --- Ligation of 4Cysl-SUMO into expression vector pET-28m and pHM6 --- p.191 / Chapter 3.2.1.5 --- Restriction digestion of pET-28m-SUMO and pHM6-SUMO for ligation with 4Cys2 oligos --- p.192 / Chapter 3.2.1.6 --- Ligation of 4Cys2 oligos to the digested pET-28m-SUMO and pHM6-SUMO plasmids --- p.193 / Chapter 3.2.1.6.1 --- Self-annealing of the 4Cys oligonucleotides --- p.193 / Chapter 3.2.1.6.2 --- Phosphorylation of ds 4Cys2 oligos and ligation to the plasmids --- p.193 / Chapter 3.2.2 --- Expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1 in E.coli expression system --- p.195 / Chapter 3.2.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.196 / Chapter 3.2.4 --- In-cell labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.197 / Chapter 3.2.4.1 --- Preparation --- p.197 / Chapter 3.2.4.2 --- In-cell Lumio´ёØ labeling --- p.198 / Chapter 3.2.4.3 --- Detection and imaging of the labeled cells --- p.199 / Chapter 3.2.5 --- In-gel labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.199 / Chapter 3.2.5.1 --- Lumio´ёØ in-gel labeling --- p.199 / Chapter 3.2.5.2 --- Visualization and imaging of the labeled gel --- p.200 / Chapter a. --- UV illumination at 302 nm --- p.200 / Chapter b. --- Typhoon Trio TMLaser-scanning at 532 nm --- p.201 / Chapter 3.2.5.3 --- Detection limit of fluorescent 4Cys2-SUMO-l in SDS-PAGE --- p.201 / Chapter 3.2.5.4 --- In-gel labelling in two-dimensional electrophoresis (2-DE) --- p.202 / Chapter 3.2.5.4.1 --- Modification of equilibration buffer before SDS-PAGE --- p.202 / Chapter 3.3 --- RESULTS --- p.203 / Chapter 3.3.1 --- Adoption of old version of 4Cys-tag (4Cys 1) in SUMO study --- p.203 / Chapter 3.3.1.1 --- Construction of recombinant pET-28m-4Cys 1 -SUMO-1 and pHM6-4Cysl-SUMO-1 --- p.203 / Chapter 3.3.1.2 --- In vivo HA-4Cysl-SUMO-1 Lumio´ёØ labelling --- p.205 / Chapter 3.3.1.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.207 / Chapter 3.3.1.4 --- Expression and purification of His6-4Cysl-SUMO-1 --- p.208 / Chapter 3.3.1.5 --- Validation of 4Cys1-SUMO-1 conjugate by Lumio´ёØ in-gel labeling --- p.211 / Chapter 3.3.2 --- Adoption of a modified version of 4Cys-tag (4Cys2) in SUMO study --- p.213 / Chapter 3.3.2.1 --- Construction of recombinant pET-28m-4Cys2-SUMO-l and pHM6-4Cys2-SUMO-l --- p.213 / Chapter 3.3.2.2 --- In vivo HA-4Cys2-SUMO-l Lumio´ёØ labelling --- p.216 / Chapter 3.3.2.3 --- Expression and purification of His6-4Cys2-SUMO-1 --- p.219 / Chapter 3.3.2.4 --- Validation of 4Cys2-SUMO-l conjugate Lumio´ёØ in-gel labeling --- p.221 / Chapter 3.3.3 --- 2As-4Cys labeling in two-dimensional electrophoresis (2-DE) --- p.223 / Chapter 3.3.3.1 --- Detection limit of 4Cys2-SUMO-l in SDS-PAGE --- p.224 / Chapter 3.3.3.2 --- Lumio´ёØ labeling in 2-DE --- p.226 / Chapter 3.4 --- DISCUSSION --- p.232 / Chapter Chapter IV --- Conclusion and Future Perspectives --- p.242 / Chapter 4.1 --- Conclusion on proteomic study of SUMOylation --- p.242 / Chapter 4.2 --- Future perspectives of proteomic study of SUMOylation --- p.245 / Chapter 4.2.1 --- In vitro study --- p.245 / Chapter 4.2.2 --- In vivo study --- p.246 / Chapter 4.3 --- Conclusion of the investigation of biarsencial-tetracysteine (2As-4Cys) system application on SUMO study --- p.247 / Chapter 4.4 --- Future perspectives of the application of 2As-4Cys system application on SUMO study --- p.249 / Chapter 4.4.1 --- In cell study --- p.249 / Chapter 4.4.2 --- In gel study --- p.250 / Appendices --- p.251 / Chapter 1. --- Genotype of E.coli strains --- p.251 / Chapter 2. --- Vector maps --- p.252 / Chapter a. --- Vector map and MCS of pET-28a --- p.252 / Chapter b. --- Vector map and MCS of pHM6 --- p.253 / Chapter c. --- Vector information of pTwo-E --- p.254 / Chapter 3. --- Primers used in this study --- p.255 / Chapter 4. --- Nikon TE2000 filter sets spectrums --- p.257 / Chapter a. --- FITC/GFP filter set --- p.257 / Chapter b. --- RFP filter set --- p.257 / Chapter c. --- UV/DAPI/Hoechst filter set --- p.258 / Chapter 5. --- Akt signalling pathway diagram --- p.259 / Chapter 6. --- DNA sequence of SUMOs and 4Cys2 oligonucleotide --- p.260 / Chapter 7. --- Electrophoresis markers --- p.261 / References --- p.263

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La protéine kinase activée par AMP (AMPK) est un senseur et régulateur central de l'état énergétique cellulaire, mais ces voies de signalisation ne sont pour le moment que partiellement comprises. Deux criblages non-biaisés pour la recherche de partenaires d'interaction et de substrats d'AMPK ont précédemment été réalisés dans le laboratoire. Ces derniers ont permis l'identification de plusieurs candidats (protéines), mais leur rôle fonctionnel et physiologique n'était pas encore établi. Ici nous avons caractérisé la fonction de la relation entre AMPK et quatre partenaires d'interaction : gluthation S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), l'E3 ubiquitine-ligase (NRDP1), et les protéines associées à la membrane (VAMP2 and VAMP3). Chacune de ces interactions parait avoir un rôle différent dans la signalisation AMPK, agissant en amont ou en aval de la protéine AMPK. GSTP1 et GSTM1 contribueraient à l'activation d'AMPK en facilitant la S-glutathionylation d'AMPK en conditions oxydatives moyennes. Cette régulation non-canonique suggère que l'AMPK peut être un senseur de l'état redox cellulaire. FH mitochondrial est l'unique substrat AMPK clairement identifié. Etonnamment le site de phosphorylation se trouve dans le peptide signal mitochondrial, ce qui pourrait affecter l'import mitochondrial. NRDP1, protéine pour laquelle nous avons pour la première fois développé un protocole de production de la protéine soluble, est faiblement phosphorylée par l'AMPK. L'interaction ne sert pas à l'ubiquitination d'AMPK, mais affecte le renouvellement de NRDP1. Finalement, l'interaction de VAMP2/3 avec AMPK n'implique pas d'évènement de phosphorylation ou d'activation d'un des partenaires. Nous proposons un mécanisme de recrutement d'AMPK par VAMP2/3 (" scaffold ") au niveau des vésicules en exocytose. Ce recrutement favoriserait la phosphorylation de substrats de l'AMPK à la surface des vésicules en exocytoses. Une fois mis en commun, nos résultats enrichissent les connaissances sur les voies de signalisation AMPK, et suggèrent une grande complexité de ces dernières. Plus que les kinases en amont et des substrats en aval, la régulation de la signalisation d'AMPK se fait via des modifications secondaires autres que la phosphorylation, via des effets sur le renouvellement de protéines, et probablement via un recrutement spécifique de l'AMPK dans certains compartiments cellulaires. / AMP-activated protein kinase (AMPK) is a central energy sensor and regulator of cellular energy state, but the AMPK signaling network is still incompletely understood. Two earlier non-biased screens for AMPK interaction partners and substrates performed in the laboratory identified several candidate proteins, but functional and physiological roles remained unclear. Here we characterized the functional relationship of AMPK with four different protein interaction partners: gluthatione S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), an E3 ubiquitin-ligase (NRDP1), and vesicle-associated membrane proteins (VAMP2 and VAMP3). Each of these interaction partners seems to have a different function in AMPK signaling, either acting up- or down-stream of AMPK. GSTP1 and GSTM1 can contribute to AMPK activation by facilitating S-glutathionylation of AMPK under mildly oxidative conditions. This non-canonical regulation suggests AMPK as a sensor of cellular redox state. Mitochondrial FH was identified as the only clear AMPK downstream substrate, but surprisingly the phosphorylation site is present in the mitochondrial targeting prepeptide, possibly affecting mitochondrial import. NRDP1, whose expression as a full-length soluble protein was achieved here for the first time, is phosphorylated by AMPK only at low levels. The interaction does neither serve for AMPK ubiquitinylation, but rather affects NRDP1 turnover. Finally, interaction of VAMP2/3 with AMPK does not involve phosphorylation or activation events of one of the partners. Instead, we propose VAMP2/3 as scaffolding proteins that recruit AMPK to exocytotic vesicles which could favor phosphorylation of vesicular AMPK substrates for exocytosis. Collectively, our results add some new elements to the AMPK signaling network, suggesting that it is much more complex than anticipated. In addition to upstream kinases and downstream substrates, regulation of AMPK signaling occurs by second

Structuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66. Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds. Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property.