Sss1p is an essential component, along with Sec61p, of the protein conducting channel (PCC) in the ER of the yeast Saccharomyces cerevisiae. It belongs to a family of proteins termed tail-anchor (TA) proteins. The TA consists of a single hydrophobic sequence at the carboxyl-terminus which anchors the protein to the membrane in a Type II (N_cytoplasm-C_lumen) orientation. TA proteins are targeted to their membranes of function through an uncharacterized SRP-independent, post-translational mechanism. The targeting mechanism and function of Sss1p are not known. In this thesis, results will be presented from targeting and functional studies of Sss1p. Sss1p is predicted to contain an ER targeting signal similar to mammalian VAMP-1A. Disruption of this putative signal caused incomplete mislocalization of Sss1p to the mitochondria which did not affect yeast growth. Mutations in the TA of Sss1p had numerous effects. Yeast expressing these mutants showed diminished growth, a defect in co-and post-translational translocation, inefficient ribosome binding to Sec61 p and the mislocalization of translocon components from light membranes (predominantly ER) to heavy membranes (predominantly mitochondria). It is argued that mutations in the TA of Sss1p disrupt the function of the protein, subsequently leading to the general defects listed above. Two possible functions for Sss1p are proposed: Sss1p is involved in forming the signal sequence binding pocket of the translocon and/or is essential for the integrity of the PCC. / Thesis / Master of Science (MS)

CONTEXTE: Au cours de l'infarctus du myocarde, l'homéostasie du calcium entre leréticulum sarcoplasmique (SR), les mitochondries et le cytosol est altérée chez lescardiomyocytes (CM) et conduit à la mort cellulaire. Les canaux de fuite de calcium sontconsidérés comme des régulateurs clés de l'homéostasie calcique réticulaire. Le translocon(TLC), un composant majeur de la machine de la traduction protéique, est un important canalde fuite calcique réticulaire.METHODES: Par des moyens optiques, nous avons d'abord évalué l'organisation spatiale etla fonction du TLC dans le SR de souris adultes CM. Dans un second temps, nous avonsinterrogé si et comment la modulation pharmacologique TLC pourrait réduire les lésionsd'ischémie/reperfusion (I/R) cardiaque dans un modèle d'infarctus du myocarde de souris.RÉSULTATS: Nos données montrent que le TLC est spécifiquement localisée dans le SRlongitudinale des CM chez la souris adulte. Nous démontrons que la puromycine (activateurpharmacologique du TLC) induit une réduction partielle des réserves de calcium dans le SRlongitudinale, alors que nous n'observons aucune altération des réserves de calcium dépendantdu récepteur ryanodine dans le SR jonctionnelle. Le préconditionnement de la souris par lapuromycine, soumis à un infarctus du myocarde, diminue significativement la zone d'infarctusde près de 30,9±6,3%. Ceci est corrélé à une diminution de l'activation des protéines proapoptotiquesmitochondriales et à une augmentation d'un mécanisme de pro-survie:l'autophagie. Nous avons également démontré que le préconditionnement de la puromycinediminue la vitesse d'augmentation du calcium dans le cytosol du CM adulte pendant la duréede l'ischémie en corrélation avec la diminution de l'activation des calpains calciques.CONCLUSIONS: Dans cette étude, nous avons caractérisé le TLC comme un canal de fuitespécifiquement situé dans le compartiment longitudinale du SR dans les CM de souris adultes.Nous avons constaté que l'activation pharmacologique de la TLC avant l'infarctus dumyocarde exerce un effet de préconditionnement sur le myocarde sans altérer les réserves de calcium dépendant de la ryanodine. Dans l'ensemble, ces résultats mettent l'accent sur les connaissances actuelles sur la dualité entre le SR jonctionnelle et le SR longitudinale et ouvrent de nouvelles perspectives thérapeutiques / BACKGROUND: During myocardial infarction, alteration of calcium homeostasis between sarcoplasmic reticulum (SR), mitochondria and cytosol occurs in cardiomyocytes (CM) and leads to cell death. Calcium leak channels are thought to be key regulators of the reticular calcium homeostasis. Translocon (TLC), a major component of the translation machinery, is a major reticular calcium leak channel.METHODS: By the mean of photonics, we first assessed the spatial organization and the function of TLC in the SR of adult mouse CM. In a second time, we questioned if and how the pharmacological TLC modulation could reduce ischemia/reperfusion (I/R)-mediated heart injury in a model of mouse myocardial infarction.RESULTS: Our data show that TLC is specifically located in the longitudinal SR in adult mouse CM. We demonstrate that puromycin induces a partial reduction of calcium stores in the longitudinal SR, while we observe no alteration in the ryanodine receptor-dependent calcium stores in the junctional SR. Puromycin preconditioning of mouse subjected to myocardial infarction significantly decreases the infarct area by near 30.9±6.3%. This is correlated with a decrease in the activation of mitochondrial pro-apoptotic proteins and an increase of a pro-survival mechanism: autophagy. We further demonstrated that puromycin preconditioning decreases the rate of calcium increase in the cytosol of adult CM during the ischemia duration in correlation with the decreased activation of calcium-dependent calpains.CONCLUSIONS: In this study, we characterized TLC as a leak channel specifically located in the longitudinal SR compartment of adult mouse CM. We found that the pharmacological activation of TLC before myocardial infarction exerts a preconditioning effect on myocardium without altering the ryanodine-dependent calcium stores. Altogether, these findings emphasize the present knowledge on the duality between junctional and longitudinal SR in CM and open up new therapeutic perspectives

Im Rahmen der vorliegenden Arbeit wurde eine mögliche Beteiligung der Protein-Translokase des Endoplasmatischen Retikulums aus Canis familiaris an der Vermittlung eines passiven Calcium-Ausstromes aus dem ER-Lumen untersucht. Der Sec61-Komplex konstituiert eine ionenpermeable Pore im Translokon des Endoplasmatischen Retikulums. Der Kanal zeigt eine hohe Dynamik im Schaltverhalten mit einer Vielzahl von Unterleitwerten, deren Mittelwerte gut mit publizierten Daten übereinstimmen. Zudem besitzt die Pore eine geringe Anionenselektivität in Experimenten mit KCl-Lösungen. Unter Verwendung von CaCl2- und MgCl2-Elektrolyten steigt diese deutlich an, was in vivo den Sec61-vermittelten, passiven Calcium-Ausstrom aus dem ER limitieren kann. Calmodulin (CaM) konnte im Rahmen der vorliegenden Arbeit als potenter Effektor des Sec61-Kanales identifiziert werden. Das Protein vermittelt ein Calcium-abhängiges, nahezu vollständiges Schließen des Kanals, während Calcium-freies ApoCalmodulin keinen Effekt auf den Offenzustand hat. Mittels Fluoreszenz-Korrelations-Spektroskopie konnte gezeigt werden, dass ein IQ-Motiv als putative Calmodulin-Bindestelle im cytosolischen N-Terminus der Sec61alpha-Untereinheit Ca2 -CaM mit nanomolarer Affinität bindet, eine Interaktion mit ApoCaM hingegen erst bei wesentlich höheren Konzentrationen stattfindet. Die CaM-vermittelte, negative Feedback-Regulation des Sec61-Komplexes durch Calcium legt einen CDI- (calcium-dependent inactivation) Mechanismus nahe, der die Membranbarriere des Endoplasmatischen Retikulums auch in Anwesenheit des weiten Translokonkanals aufrecht erhält.Vergleichende Experimente haben zudem ergeben, dass der Sec61-Kanal aus Saccharomyces cerevisiae im Hinblick auf die grundsätzlichen elektrophysiologischen Eigenschaften übereinstimmende Charakteristika mit dem Komplex aus Canis familiaris zeigt.

<p>  </p> <p>Protein translocation across the chloroplast outer membrane is essential for photosynthesis in all plants and certain algae. This is because most chloroplast proteins (over 90%) are encoded in the nucleus, translated in the cytoplasm, and must be imported into the chloroplasts to perform their function. The translocon at the outer chloroplast membrane (TOC) complex orchestrates this vital translocation process and consists of three components in plants: Toc75, Toc33/34 and Toc159. Our overall goal is to elucidate the architecture of the TOC complex to gain mechanistic insights into protein translocation into chloroplasts. However, the major bottleneck preventing structure determination of the TOC complex has been the inability to produce or isolate the complex to sufficient yields and purity for structural studies. We began by using phage display to screen for synthetic antigen-binding fragments (sABs) that bind to the soluble POTRA domains of Toc75 from both <em>Arabidopsis thaliana</em> and <em>Pisum sativum</em>. We then characterized the POTRA-sAB interactions using size-exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS), isothermal titration calorimetry (ITC), and X-ray crystallography. Finally, we show that we can use an affinity tagged sAB to isolate the TOC complex directly from pea biomass. This study has paved the way for high-resolution structural studies of the TOC complex from plants to understand protein translocation mechanisms. </p>

Type III secretion systems (T3SS) are a common feature of Gram-negative bacteria, allowing them to inject anti-host effectors into the interior of infected eukaryotic cells. By this mechanism, these virulence factors help the bacteria to modulate eukaryotic cell function in its favor and subvert host innate immunity. This promotes a less hostile environment in which infecting bacteria can colonize and cause disease. In pathogenic Yersinia, a crucial protein in this process is YopD. YopD is a T3S substrate that, together with YopB, forms a translocon pore in the host cell membrane through which the Yop effectors may gain access to the target-cell cytosol. The assembly of the translocator pore in plasma membranes is considered a fundamental feature of all T3SSs. How the pore is formed, what determines the correct size and ultimately the stoichiometry between YopD YopB, is still unknown. Portions of YopD are also observed inside HeLa cells. Moreover, YopD functions together with its T3S chaperone, LcrH, to control Yops synthesis in the bacterial cytoplasm. The multifunctional YopD may influence all these processes by compartmentalizing activities into discrete modular domains along the protein length. Therefore, understanding how particular domains and/or residues within these regions coordinate multiple functions of the protein will provide a platform to improve our knowledge of the molecular mechanisms behind translocation through T3SSs. Comprehensive site-directed mutagenesis of the YopD C-terminal amphipathic α-helix domain, pinpointed hydrophobic residues as important for YopD function. Some YopD variants were defective in self-assembly and in the ability to interact with the needle tip protein, LcrV, which were required to facilitate bacterial T3S activity. A similar mutagenesis approach was used to understand the role of the two predicted coiled-coils located at the N-terminal and C-terminal region of YopD. The predicted N-terminal element that occurs solely in the Yersinia YopD translocator family is essential for optimal T3SS and full disease progression. The predicted YopD C-terminal coiled-coil shapes a functional translocon inserted into host cell membranes. This translocon was seen to be a dynamic structure facilitating at least two roles during effectors delivery into cells; one to guarantee translocon pore insertion into target cell membranes and the other to promote targeted activity of internalized effector toxins. In Yersinia expression of yop genes and secretion of the corresponding polypeptides is tightly regulated at a transcriptional and post-transcriptional level. If T3S chaperones of the translocator class are known to influence transcriptional output of T3SS genes in other bacteria, we show that in Yersinia the class II T3S chaperone LcrH has no such effect on the LcrF transcriptional activator activity. We also demonstrate that there are possibly additional yop-regulatory roles for the LcrH chaperone besides forming a stable complex with YopD to impose post-transcriptional silencing on Yops synthesis. This mechanism that relies upon an active T3SS, might act independently of both YopD and the regulatory element LcrQ. In conclusion, this work has sought to delineate the encrypted functions of the YopD translocator that contribute to Yersinia T3SS-dependent pathogenesis. Contributions of the YopD cognate chaperone LcrH in yop regulatory control are also presented.

Membrane proteins comprise around 20-30% of a typical proteome and play crucial roles in a wide variety of biochemical pathways. Apart from their general biological significance, membrane proteins are of particular interest to the pharmaceutical industry, being targets for more than half of all available drugs. This thesis focuses on prediction methods for membrane proteins that ultimately rely on their amino acid sequence only. By identifying soluble protein domains in membrane protein sequences, we were able to constrain and improve prediction of membrane protein topology, i.e. what parts of the sequence span the membrane and what parts are located on the cytoplasmic and extra-cytoplasmic sides. Using predicted topology as input to a profile-profile based alignment protocol, we managed to increase sensitivity to detect distant membrane protein homologs. Finally, experimental measurements of the level of membrane integration of systematically designed transmembrane helices in vitro were used to derive a scale of position-specific contributions to helix insertion efficiency for all 20 naturally occurring amino acids. Notably, position within the helix was found to be an important factor for the contribution to helix insertion efficiency for polar and charged amino acids, reflecting the highly anisotropic environment of the membrane. Using the scale to predict natural transmembrane helices in protein sequences revealed that, whereas helices in single-spanning proteins are typically hydrophobic enough to insert by themselves, a large part of the helices in multi-spanning proteins seem to require stabilizing helix-helix interactions for proper membrane integration. Implementing the scale to predict full transmembrane topologies yielded results comparable to the best statistics-based topology prediction methods.

alpha-Helical membrane proteins are important for many biological functions. Due to physicochemical constraints, the structures of membrane proteins differ from the structure of soluble proteins. Historically, membrane protein structures were assumed to be more or less two-dimensional, consisting of long, straight, membrane-spanning parallel helices packed against each other. However, during the past decade, a number of the new membrane protein structures cast doubt on this notion. Today, it is evident that the structures of many membrane proteins are equally complex as for many soluble proteins. Here, we review this development and discuss the consequences for our understanding of membrane protein biogenesis, folding, evolution, and bioinformatics. / <p>authorCount :3</p>

Chez la bactérie Escherichia coli, SecB appartient au réseau de chaperons moléculaires qui assistent le repliement et l'adressage des protéines nouvellement synthétisées. SecB est connu pour faciliter l'export en interagissant avec les pré-protéines sous forme non-native et les adressant au translocon Sec via une interaction directe avec le moteur ATPase SecA. SecB possède aussi une activité de chaperon générique et est capable d'assister le repliement de certaines protéines cytosoliques en absence des chaperons majeurs DnaK et Trigger factor. Alors que le translocon Sec est universellement conservé, le chaperon SecB est retrouvé principalement chez les protéobactéries. Cependant, de plus en plus de séquences de type SecB sont retrouvées dans d'autres groupes de la taxonomie bactérienne, comme par exemple chez le pathogène humain majeur Mycobacterium tuberculosis. Chez cette bactérie, une séquence de type SecB appelée Rv1957 est présente en association avec un système toxine-antitoxin (TA) appartenant à la famille HigBA. Généralement, les TA sont des systèmes à deux composants qui modulent la croissance en réponse à des conditions de stress spécifiques, favorisant ainsi l'adaptation et la persistance. Dans le cas de ce système atypique toxine-antitoxine-chaperon (TAC), Rv1957 interagit avec l'antitoxine HigA et la protège à la fois de l'agrégation et de la dégradation, et est donc strictement requis pour permettre l'inactivation de la toxine par l'antitoxine. La première partie de ce travail avait pour but de reconstruire l'histoire évolutive de ce nouveau système TAC. Pour cela nous avons procédé à une recherche de systèmes similaires dans l'ensemble des génomes disponibles qui a révélé que la présence de systèmes TAC n'est pas limitée aux mycobactéries et que ces systèmes semblent s'être répandus dans la taxonomie par le biais de transferts horizontaux de gènes. Nos résultats suggèrent que les chaperons des systèmes TAC sont évolutivement apparentés au chaperon d'export solitaire SecB et ont divergé pour devenir spécialisés vis-à-vis de leurs antitoxines partenaires. Nous avons ensuite étudié ce phénomène de spécialisation par une approche d'évolution dirigée du chaperon d'export SecB d'E. coli. Nous avons mis en évidence que des substitutions uniques dans SecB sont suffisantes pour améliorer sa capacité à contrôler spécifiquement HigBA du système TAC, et que ces mutations résultaient généralement en une meilleure interaction avec l'antitoxine HigA. Remarquablement, environ la moitié des mutants identifiés sont affectés dans leur activité de chaperon générique en l'absence des chaperons DnaK et Trigger factor, suggérant un conflit entre spécialisation du chaperon et ses fonctions génériques. La plupart des résidus identifiés se trouvent dans une région de SecB non caractérisée et proche du site proposé d'interaction avec le substrat. Des expériences de cross-link à des positions spécifiques ont révélé que cette région interagit directement avec l'antitoxine HigA. Enfin, nous avons montré que HigA est capable d'entrer en compétition avec la fonction d'export d'un SecB spécialisé plus efficacement que pour la version sauvage de SecB, illustrant la potentielle connexion entre les fonctions de type SecB dans l'export et le contrôle d'un système TA. / SecB is part of the intricate network of chaperones that assist folding and targeting of newly synthesized polypeptides in Escherichia coli. SecB is known to interact with nonnative precursor proteins and address them to the Sec translocon via direct interaction with the SecA motor component, thus facilitating their export. SecB is also able to act as a generic chaperone, by assisting the folding of certain cytosolic proteins when the major DnaK/Trigger Factor chaperone pathway is disrupted. While the Sec translocon is universally conserved, the SecB chaperone is mainly found in proteobacteria. However, an increasing number of SecB-like sequences have been found in unusual groups of bacteria and especially in the major human pathogen Mycobacterium tuberculosis. In this bacterium, a SecB-like sequence, Rv1957, is present in association with a toxin-antitoxin (TA) system belonging to the HigBA family. Usually, TA modules are two-component systems that modulate growth in response to specific stress conditions, thus promoting adaptation and persistence. In the case of this atypical toxin-antitoxin-chaperone (TAC) system, Rv1957 interacts with the HigA antitoxin and protects it from both aggregation and degradation, and is thus strictly required for neutralization of the toxin by the antitoxin. The first aim of this work was to reconstruct the evolutionary history of the newly discovered TAC system. We performed a large-scale genome screening and found that TAC is not restricted to mycobacteria and seems to have disseminated in the taxonomy by horizontal gene transfer. Our results suggest that TAC chaperones are evolutionarily related to the solitary export chaperone SecB and have diverged to become specialized towards their cognate antitoxins. Next, we investigated such chaperone specialization event through directed evolution of the E. coli export chaperone SecB. We found that single amino-acid substitutions within SecB were sufficient to improve its ability to specifically control HigBA from TAC, and that these mutations mainly resulted in an increased binding to the HigA antitoxin. Strikingly, about half of the mutants identified were affected in their ability to perform SecB generic chaperone functions in the absence of both DnaK and TF chaperones, suggesting a conflict between specialization and generic chaperone functions. Most of the residues identified are located within a previously uncharacterized region of SecB which is close to the proposed substrate binding site. Further in vitro site-specific cross-linking experiments revealed that this region directly interacts with the HigA antitoxin. Finally, we show that the HigA antitoxin can compete with the export function of specialized SecB more efficiently than it does with wild type SecB, thus illustrating the potential interplay between SecB-like chaperone export functions and TA activation.

No description available.

Le diabète de type 2 est une pathologie chronique complexe associant une altération de sécrétion de l'insuline par le pancréas et une résistance à l'insuline au niveau des tissus périphériques, notamment au niveau du foie et du muscle squelettique. Son origine est multifactorielle, alliant des anomalies génétiques et environnementales, en particulier nutritionnelles. Un des mécanismes par lesquels les facteurs nutritionnels (comme les glucides et les lipides en excès) contribuent au développement du diabète et à son aggravation est la glucolipotoxicité. En effet, l'élévation de la glycémie et des lipides plasmatiques, ainsi que l'accumulation ectopique de lipides dans les tissus, participent au développement de l'insulinorésistance hépatique et musculaire et aux dysfonctions des cellules bêta, en partie via l'induction d'un stress métabolique, impliquant notamment le stress oxydant, le stress du réticulum endoplasmique (RE) et la perturbation de l'homéostasie calcique. Nous avons étudié les effets de la glucotoxicité et de la lipotoxicité dans les cellules bêta pancréatiques et les mécanismes impliqués. Nous nous sommes aussi intéressés à des traitements potentiellement protecteurs de la fonction bêta-pancréatique. Nous avons fait l'hypothèse que les effets bénéfiques de l'inhibition du système rénine-angiotensine sur l'incidence du diabète de type 2 chez l'homme étaient médiés par une action directe sur les cellules bêta. Nos résultats montrent que le glucose chronique à une dose élevée entraine une réduction de la sécrétion d'insuline des cellules bêta des îlots de Langerhans humains par une action conjointe sur le stress du RE, le stress oxydant et l'homéostasie calcique. L'inhibition du SRA a permis de restaurer cette fonction grâce notamment à une action inhibitrice sur la voie Phospholipase C-IP3-Calcium / This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in type 2 diabetes, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor t- AUCB (10 mg/l in drinking water) were compared to those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 weeks in FVB mice subjected to a high-fat diet (HFD, 60% fat) for 16 weeks. Mice on control chow diet (10% fat) and non-treated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids and LDL cholesterol, and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the NO-synthase and cytochrome P450 epoxygenase inhibitors, L-NA and MSPPOH, on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis and inflammation, and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the enddiastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that she inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese type 2 diabetic mice