The central role played by the Endoplasmic Reticulum (ER) in the correct folding and assembly of secretary and membrane proteins cannot be overstated. As the first compartment in the secretary pathway, it is responsible for the synthesis, modification and targeting of proteins to their proper destinations within the secretary pathway and the extracellular space. Protein folding in this specialized compartment is dynamic and involves a host of molecular chaperones and folding catalysts. Once inside the ER lumen, proteins fold into their native conformation and undergo a multitude of post-translational modifications, including N-linked glycosylation and disulfide oxidation. The proper conformational maturation of nascent proteins that traverse the secretary pathway is both aided and monitored by a complex process termed ER quality control. A variety of quality control mechanisms that rely on the chaperone systems operate in the ER. These act in close concert with the molecular machinery involved in degradation of non-native proteins to maintain homeostasis. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. As a general rule, only those proteins that have successfully completed their folding and passed a stringent selection process are allowed to exit the ER on their way to their final destinations. The importance of the normal functioning of the ER is underlined by the fact that disruption in protein folding, resulting in ER stress, has now been identified as the biochemical basis of many ER storage diseases including Diabetes mellitus, Endocrinopathies and Hemophilia A. Processing events occurring inside the ER lumen are known to influence the efficiency of protein secretion. Vastly different rates of exocytose observed among secretary proteins have been found to correlate with the rate of exit from the ER. One such example is the interesting secretion property exhibited by Retinol Binding Protein (RBP) The principal carrier of retinol (Vitamin A) in plasma. RBP is a single domain protein consisting of three intramolecular disulfide bonds and helps transport retinol from the liver stores to the various target tissues in the body. Availability of its ligand, retinol, while not affecting its synthesis, is known to be the major factor in regulating RBP secretion from the liver. In the absence of retinol, apo-RBP has been shown to be retained in the ER by a hitherto unclear mechanism. Like most other secretary proteins, RBP is co-translationally targeted to the ER lumen, where it undergoes disulfide oxidation as the only modification. It has been shown to form a complex with another secretary protein, Transthyretin (TTR) in the ER and this complex formation is thought to prevent premature glomerular filtration of the otherwise small RBP with its bound retinol. Despite attaining a mature conformation, apo-RBP is not secreted and awaits conversion to its ligand-bound, holo form in order to exit the ER. It is widely believed that ligand binding may relieve this retention of RBP from the ER quality control machinery. However the precise mechanisms that mediate and regulate RBP folding, ligand binding, TTR assembly and secretion are not clearly understood. Though the folding and secretion properties of RBP have been described in HepG2 cells, its interactions with the ER resident chaperones have not been addressed. Apart from being an important cell biological question, the study of RBP assumes a lot of significance with its recent emergence as a key player in the pathogenesis of type 2 diabetes mellitus. It has been proposed that lowering of serum RBP levels could be a new strategy for treating type 2 diabetes mellitus. The present study was undertaken with the intention of analyzing the oxidative folding of RBP in the ER more closely. A systematic approach aimed at understanding the early events associated with folding and maturation of RBP, with particular emphasis on the role of ER-resident chaperones and the quality control machinery, is likely to provide interesting insights into the mechanisms involved in its ligand dependent secretion. Reconstitution of RBP biogenesis in a cell free system. The folding of RBP in cells is extremely quick with rapid oxidation kinetics. This makes it difficult to systematically analyze the early folding events in cultured cells. It was necessary to make use of a simplified system that would faithfully recapitulate the folding process in the ER. Therefore, a cell free translation system consisting of rabbit reticulocyte lysate and canine pancreatic microcosms as a source of ER-derived membranes was developed. This system affords the advantage of easy manipulation while still preserving the overall environment that prevails in the ER of intact cells. Extensive biochemical and functional characterization of the isolated microcosms was carried out and in vitro translation and microsomal translocation of RBP was established. Though initially confined to studies on membrane insertion and core glycosylate, the cell free system supplemented with microcosms has subsequently been used to analyze folding and assembly of a number of secretary and membrane proteins. A similar strategy has been adopted in the present study of RBP folding and maturation. Oxidative folding of RBP in isolated microcosms: Delineation of its disulfide oxidation pathway Using glutathione (GSSG) as the oxidant, co- and posttranslational disulfide oxidation of RBP was carried out in isolated microcosms. The ability to manipulate the redox status of this cell free system has helped to considerably slow down the oxidative folding of RBP so that a more careful analysis of the folding process could be performed. RBP was found to undergo oxidative folding with a t1/2 of 30 minutes and folding proceeded through at least one disulfide-bonded intermediate. Non-reducing SDS PAGE was used to resolve the folding intermediates. The pattern of oxidation was in good agreement with that reported earlier in HepG2 cells. No significant effect of retinol was observed on either the folding kinetics or the pattern of disulfide oxidation of RBP in isolated microsomes.A DTT sensitivity assay, used to probe the conformational maturity of folding RBP, revealed that RBP was capable of maturing into a DTT-resistant conformation in isolated microsomes. With the aid of disulfide mutants, the probable disulfide oxidation pathway of RBP in the ER has been determined. Single and double disulfide mutants of RBP were generated by site-directed mutagenesis and their posttranslational oxidation patterns were analyzed and compared with that of the wild type protein. Based on the results obtained, it was clear that the folding intermediate was made up of one of the two big disulfide loops and that the presence of both these loops was essential for RBP to fold into a fully oxidized, compact form. It has not been possible to determine the contribution of the third, smallest disulfide loop to the oxidative folding of RBP. Molecular events associated with the early oxidative folding of RBP To gain insights into the possible role of ER chaperones in the oxidative folding of RBP, the oligomeric state of folding RBP was analyzed by velocity sedimentation and chemical crosslinking assays. Velocity sedimentation analysis revealed that the reduced form of RBP was present in a large complex of size >100 S20,W. Upon disulfide oxidation, it readily dissociated from the complex and assumed a monomeric state. This was evident even during co-translational oxidation which suggested that RBP transiently associated with the large complex during its oxidative folding. Dynamic nature of this complex indicated that this could be a folding complex containing the chaperone machinery of the ER. These results were also supported by crosslinking analysis performed in unbroken microsomes using the homo-bifunctional crosslinker, DSP. The early folding forms of RBP could be crosslinked to a large complex while upon disulfide oxidation, RBP matured to its monomeric form and was no longer crosslinkable. Sedimentation and crosslinking analyses of the RBP disulfide mutants revealed that while the double disulfide mutant remained irreversibly associated with the large complex, the single mutants were released upon acquiring one of the two big disulfide loops. This suggested that despite the lack of one of the two major disulfides, these mutants were considered ‘folded’ by the quality control machinery in the ER while the double mutant probably resembled a molten globule state and was therefore considered ‘unfolded’ and irreversibly retained. Results from crosslinking analysis in microsomes not engaged in active translation suggested that chaperones of the ER were organized in a complex constitutively thereby lending support to the concept of ER-matrix, a large network of luminal proteins consisting of ER chaperones and accessory factors. Given this scenario, it is not unlikely that newly synthesized protein substrates transiently associate with this large pre-existing complex of chaperones and dissociate during late stages of their maturation. Conclusion In all, this study provides significant insights into some of the early events associated with the oxidative folding of RBP in the ER. The delineation of the disulfide oxidation pathway of RBP has been possible. The results obtained from this study suggest that RBP probably dissociates from the quality control quite early during its folding process and this step in its maturation might not be influenced by retinol. The stimulus for its ligant dependent secretion is likely to operate at a later stage of its sojourn in the ER, possibly consequent to positive cues from accessory binding factors such as TTR. Lastly, Perservation of the ER microenvironment in isolated microsomes, as evidenced from this study, augurs well for the use of this system to analyze mechanisms underlying folding, maturation, secretion and/or retention of secretory proteins.

The study of RNA-binding proteins has recently increased in importance due to discoveries of their larger role in cellular processes. One study currently conducted at Umeå University involves constructing a model that will be able to improve our knowledge about T-cells by explaining how these cells work in different diseases. But before this model can become a reality, Umeå Univerity needs to investigate the relation between RNA and RNA-binding proteins and find proteins of which highly contribute to the activity of the RNA-binding proteins. To do so, they have decided to use four penalized regression Machine Learning models to analyse protein sequences from CD4 cells. These models consist of a ridge penalized model, an elastic net model, a neural network model, and a Bayesian model. The results show that the models have a number of RNA-binding protein sequences in common which they list as highly decisive in their predictions.

No description available.

La dystrophie myotonique de type 1 (DM1) est une maladie dégénérative impliquant des symptômes d’atrophie musculaire et de myotonie. Au niveau moléculaire, elle est caractérisée par une expansion aberrante de CUG dans la région 3’UTR de l’ARNm de DMPK (Dystrophia Myotonica protein kinase). Ces répétitions CUG forment des agrégats toxiques (appelés foci) majoritairement nucléaires dans les cellules de patients DM1 et causent la séquestration anormale de ribonucléoprotéines (RBP), tel que le facteur «Muscleblind-like 1» (MBNL1), qui lieraient normalement les motifs CUG d’autres ARN. Les fonctions normales de ces RBPs seraient alors perturbées. En plus de leur rôle dans l’épissage alternatif, MBNL a récemment été caractérisé pour son implication dans la localisation intracellulaire de ses ARN cibles. Ceci suggèrerait que la pathogénèse de la DM1 pourrait résulter de l’effet perturbateur des répétitions CUG sur la localisation d’ARN précis et de protéines RBPs. À cet effet, un criblage basé sur de la microscopie fluorescente de 322 RBPs dans des myoblastes de patients DM1 a permis d’identifier des nouveaux facteurs qui colocaliseraient avec les expansions pathogéniques CUG. De plus, ces myoblastes DM1 ont été fractionnés et un séquençage-ARN a par la suite permis l’identification de transcrits délocalisés. Les deux banques de données ainsi générées, tant par le criblage que par le fractionnement/séquençage-ARN, pourraient ouvrir des nouvelles avenues de recherches dans la compréhension des anomalies moléculaires associées à la DM1, et potentiellement d’autres maladies à expansions microsatellites. / Myotonic dystrophy of type 1 (DM1) is a degenerative disorder implicating symptoms of muscular atrophy and myotony. In a molecular level, it is caused by the aberrant expansion of CUG repeats in the 3’-UTR region of the DMPK mRNA (Dystrophia Myotonica protein kinase). Excessive CUG repeats then form toxic aggregates (foci) enriched within the nucleus of DM1 patient cells. These RNA foci cause the abnormal sequestration of RNA Binding Proteins (RBP), in particular members of the Muscleblind-like protein 1 (MBNL), that normally bind the CUG motif of other target RNAs, and will hence alter their normal functions. In addition to their role in alternative splicing, MBNL1 has recently been implicated in the intracellular localisation of its RNA targets. It remains elusive whether the pathogenesis of DM1 could result from the deregulating effect of CUG repeats on the localisation of specific RNAs and RBP proteins. In this thesis, a fluorescent imaging-based screening of 322 RBPs in DM1 patient’s myoblasts has been conducted and this had led to the identification of new factors that may colocalize with pathogenic CUG expansions. Moreover, these DM1 myoblasts have been fractionated and subsequent RNA-sequencing has permitted the identification of transcripts that are delocalised between subcellular compartments. From the two large datasets generated from the RBP imaging-based screening and fractionation/RNA-sequencing, new avenues of research can be initiated to further understand not only DM1, but perhaps also other disorders that implicate microsatellite expansions.

Indiana University-Purdue University Indianapolis (IUPUI) / My thesis comprises of two individual projects which revolve around the importance of post-transcriptional regulation in liver. My first project is studying the integrated miRNA – mRNA network in NAFLD. For fulfillment of the study we conducted a genome-wide study to identify microRNAs (miRs) as well as the miR-mRNA regulatory network associated with hepatic fat and NAFLD. Hepatic fat content (HFC), miR and mRNA expression were assessed in 73 human liver samples. Liver histology of 49 samples was further characterized into normal (n=33) and NAFLD (n=16). Liver miRNome and transcriptome were significantly associated with HFC and utilized to (a) build miR-mRNA association networks in NAFLD and normal livers separately based on the potential miR-mRNA targeting and (b) conduct pathway enrichment analyses. We identified 62 miRs significantly correlated with HFC (p < 0.05 with q < 0.15), with miR-518b and miR-19b being most positively and negatively correlated with HFC, respectively (p < 0.008 for both). Integrated network analysis showed that six miRs (miRs-30b*, 612, 17*, 129-5p, 204 and 20a) controlled ~ 70% of 151 HFC-associated mRNAs (p < 0.001 with q < 0.005). Pathway analyses of these HFC-associated mRNA revealed their key effect (p<0.05) in inflammation pathways and lipid metabolism. Further, significant (p<2.47e-4, Wilcoxon test) reduction in degree of negative associations for HFC-associated miRs with HFC-associated mRNAs was observed in NAFLD as compared to normal livers, strongly suggesting highly dysfunctional miR-mRNA post-transcriptional regulatory network in NAFLD. Our study makes several novel observations which provide clues to better understand the pathogenesis and potential treatment targets of NAFLD. My second project is based on uncovering important players of post-transcriptional regulation (RBPs) and how they are associated with age and gender during healthy liver development. For this study, we performed an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations show that several of these RBPs are important developmentally conserved regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes.

Die Genexpression wurde bisher hauptsächlich auf Transkriptions- und Proteinebene untersucht, wobei der Einfluss der Translation, die die Proteinhäufigkeit direkt beeinflusst, weitgehend außer Acht gelassen wurde. Um diese Rolle besser zu verstehen, habe ich Ribosomen-Profiling-Daten (Ribo-seq) verwendet, um die Translationsregulation zu untersuchen und neue Translationsvorgänge in 65 linksventrikulären Proben von DCM-Patienten im Endstadium und 15 Nicht-DCM-Kontrollen zu identifizieren. Dieser Datensatz half dabei, die Transkriptions- und Translationsregulation zwischen erkrankten und nicht betroffenen menschlichen Herzen zu sezieren und enthüllte Gene und Prozesse, die rein unter Translationskontrolle stehen. Darüber hinaus habe ich neue kardiale Proteine vorhergesagt, die von langen nicht-kodierenden RNAs (lncRNAs) und zirkulären RNAs (circRNAs) translatiert werden. Computergestützte Analysen dieser evolutionär jungen Proteine legten eine Beteiligung an verschiedenen molekularen Prozessen nahe, mit einer besonderen Anreicherung für den mitochondrialen Energiestoffwechsel. Schließlich identifizierte ich RNA-bindende Proteine (RBPs), deren Expression die Menge der Ziel-mRNA oder die Frequenz der Translationseffizienz (TE) beeinflusst. Für eine Untergruppe von 21 RBPs habe ich die Regulation auf beiden quantitativen Merkmalen beobachtet, was zu einer unterschiedlichen mechanistischen Basis der Expressionskontrolle für unabhängige Gensätze führte. Obwohl die genaue Umschaltung der RBP-Funktion wahrscheinlich durch eine Kombination von mehreren Faktoren erreicht wird, haben wir für drei Kandidaten eine starke Abhängigkeit von der Zielgenlänge und der 5'-UTR-Struktur beobachtet. Diese Arbeit präsentiert einen Katalog von neu identifizierten Translationsereignissen und einen quantitativen Ansatz zur Untersuchung der Translationsregulation im gesunden und kranken menschlichen Herzen. / Gene expression has primarily been studied on transcriptional and protein levels, largely disregarding the extent of translational regulation that directly influences protein abundance. To elucidate its role, I used ribosome profiling (Ribo-seq) data, obtained through ribosome profiling, to study translational regulation and identify novel translation events in 65 left ventricular samples of end-stage DCM patients and 15 non-DCM controls. This dataset helped dissect transcriptional and translational regulation between diseased and unaffected human hearts, revealing genes and processes purely under translational control. These would have remained undetected by only looking at the transcriptional level. Furthermore, I predicted novel cardiac proteins translated from long non-coding RNAs (lncRNAs) and circRNAs. Computational analysis of these evolutionary young proteins suggested involvement in diverse molecular processes with a particular enrichment for mitochondrial processes. Finally, I identified RNA-binding proteins (RBPs) whose expression influences target mRNA abundance or translational efficiency (TE) rates. For a subset of 21 RBPs, I have observed regulation on both quantitative traits, which resulted in different mechanistic basis expression control for independent sets of genes. Though the precise switch in RBP function is likely achieved by a combination of multiple factors, for three candidates we have observed a strong dependency on target length and 5’ UTR structure. This work presents a catalogue of newly identified translation events and a quantitative approach to study translational regulation in the healthy and failing human heart.

Les biopuces à ADN permettent d’étudier à une échelle génomique une très grande variété de questions sur la physiologie et la différenciation cellulaires. Elles ont ainsi contribué de manière considérable aux progrès récents de nombreux domaines de la biologie et occuperont bientôt une place de choix dans le secteur du diagnostic médical. C’est la levure Saccharomyces cerevisiae qui a servi de modèle pour le développement de la première biopuce génomique. L’application de cette approche à la levure a permis d’explorer sous un angle nouveau l’étude de ses différents états de différenciation, de son cycle cellulaire, et de sa capacité d’adaptation à diverses conditions nutritionnelles ou à des conditions environnementales induisant un stress cellulaire. Plusieurs études ont plus particulièrement examiné la réponse des cellules de levure à une carence en azote ou en acides aminés. Cependant, une étude systématique de la réponse transcriptionnelle de la cellule aux différentes sources d’azote n’a jamais été entreprise en croissance confinée à l'état de régime. S. cerevisiae est capable d’utiliser plus d’une vingtaine de substances en tant que sources uniques d’azote pour la croissance. On distingue parmi les sources d’azote celles qui permettent une croissance optimale, appelées « bonnes » sources d’azote, des autres, appelées « mauvaises » sources d’azote. La levure possède plusieurs systèmes de régulation lui permettant de s'adapter à la condition azotée. Au niveau transcriptionnel, on recense trois régulations générales – la NCR (répression catabolique azotée), le GAAC (le contrôle général de la biosynthèse des acides aminés) et le système SPS (Ssy1-Ptr3-Ssy5) – et une multitude de régulations plus spécifiques.<p>En utilisant la technique des puces à ADN, nous avons généré une matrice d'expression de l'ensemble des gènes de la levure en croissance confinée à l'état de régime dans un milieu de culture contenant une parmi 21 sources d'azote différentes. Nous avons pu ainsi recenser systématiquement 506 gènes soumis à une régulation transcriptionnelle dépendante de l'azote.<p>En nous basant sur ces résultats, nous avons pu décrire l'ensemble des régulations transcriptionnelles engagées dans l'adaptation à la source d'azote fournie dans le milieu de culture. Parallèlement, nous avons défini deux grands groupes de sources d'azote en fonction du transcriptome de S. cerevisiae. Le premier groupe rassemble les composés qui exercent une répression catabolique azotée forte et dont la liste a été complétée. Fait nouveau, nous montrons que ces mêmes composés enclenchent aussi l'activation de la réponse aux protéines mal repliées (UPR). Au contraire, lorsque la source d'azote appartient au second groupe que nous avons défini, non seulement la croissance des levures est plus lente, la NCR levée et la réponse aux protéines mal repliées réprimée, mais nous montrons de façon inattendue que le contrôle général de la biosynthèse des acides aminés est activé. Plusieurs autres régulations qui ne sont pas impliquées dans le métabolisme azoté présentent un comportement différent en fonction de la source d'azote fournie. C'est le cas notamment des gènes dont l’expression est régulée selon l’apport en zinc et qui sont moins exprimés sur le milieu urée. De même, les gènes impliqués dans les résistances multiples aux drogues sont activés par le tryptophane.<p>En confrontant nos résultats à ceux obtenus dans le cadre de travaux indépendants, nous avons proposé plus d'une cinquantaine de nouveaux gènes cibles de la NCR. Beaucoup d'entre eux n'ont jamais été caractérisés expérimentalement. En utilisant des techniques avancées d'analyse de séquences primaires de protéines, nous avons pu proposer une fonction pour plusieurs de ces gènes. Ces analyses bioinformatiques et la réalisation d’expériences complémentaires à l’aide de biopuces à ADN nous ont permis de proposer que l'un d'entre eux code pour une protéine impliquée dans la déstabilisation d'ARN messagers lors de la carence azotée. Nous avons aussi identifié plusieurs nouveaux gènes appartenant à des régulons spécifiquement activés en réponse à un nombre restreint de sources d'azote. Il est probable que ces gènes soient impliqués dans le catabolisme des sources d'azote sur lesquelles ils sont activés. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished