Дисертації з теми "MFS proteins"

La ferroportine-1 (FPN1), seul exportateur de fer connu chez les mammifères, est exprimée à la surface de différentes cellules spécialisées du métabolisme de fer. Cette protéine appartient à la famille des protéines MFS « Major Facilitator Superfamily » et libère le fer intracellulaire au travers de changements conformationnels oscillant entre une structure ouverte vers le cytoplasme (« Inward-Facing ») et une structure ouverte vers la circulation sanguine (« Outward-Facing » ; OF). Il a été rapporté que FPN1 est préférentiellement localisée dans les radeaux lipidiques, des microdomaines de la membrane plasmique particulièrement enrichies en cholestérol (CHOL). Au début de la thèse nous avons formulé l’hypothèse que des interactions directes entre FPN1 et les lipides environnants, notamment le CHOL, sont nécessaires pour stabiliser FPN1 dans la conformation OF et/ou favoriser certains changements conformationnels. J’ai confirmé la colocalisation préférentielle de FPN1 dans les radeaux lipidiques des cellules embryonnaires de rein humain. La dépendance de la fonction d’export de fer de FPN1 au CHOL a été examinée par déplétion/réplétion (CHOL/épicholestérol). Des expériences de criblage mutationnel appuyées par des analyses structurelles de la structure expérimentale 3D de FPN1 dans un état OF ont permis d’identifier trois sites de liaison possibles au CHOL (de types CARC/CRAC). Sur la base de simulations de dynamique moléculaire dans un environnement lipidique simplifié de type POPC, nous identifions certaines interactions entre des résidus chargés de la structure 3D FPN1 humaine et les têtes polaires de phospholipides environnants, qui pourraient faciliter les changements conformationnels du transporteur. Je montre également, pour la première fois, que la fonction de FPN1 est modulée par des composés amphiphiles de synthèse, l’ohmline et ses dérivés. Au travers du développement d’une nouvelle approche in vitro (PLA : « Proximity Ligation Assay »), j’indique que l’ohmline délocalise FPN1 des radeaux lipidiques, diminuant ainsi son interaction avec son partenaire fonctionnel, la céruloplasmine (CP), une ferroxydase qui catalyse l'oxydation du fer ferreux, et en conséquence la fonction d’export du fer
Ferroportin-1(FPN1), the only known mammalian iron exporter, is expressed on the surface of various specialized cells involved in iron metabolism. This protein belongs to the Major Facilitator Superfamily (MFS) and releases intracellular iron through conformational changes oscillating between an open structure towards the cytoplasm (Inward-Facing) and an open structure towards the bloodstream (Outward-Facing; OF). It has been reported that FPN1 is preferentially localized in lipid-rafts, microdomains of the plasma membrane particularly enriched in cholesterol (CHOL). Early in the thesis, we hypothesized that direct interactions between FPN1 and surrounding lipids, notably CHOL, are necessary to stabilize FPN1 in the OF conformation and/or promote certain conformational changes. I confirmed the preferential colocalization of FPN1 in the lipid rafts of human embryonic kidney cells. The dependence of FPN1's iron export function on CHOL was examined by depletion/repletion (CHOL/epicholesterol). Mutational screening experiments supported by structural analyses of the experimental 3D structure of FPN1 in an OF state have identified three possible CHOL-binding sites (of the CARC/CRAC type). Based on molecular dynamics simulations in a simplified POPC-type lipid environment, we identify certain interactions between charged residues of the human FPN1 3D structure and the polar heads of the surrounding phospholipids, which could facilitate conformational changes of the transporter. Besides, I show, for the first time, that FPN1 function is modulated by synthetic amphiphilic compounds, ohmline and its derivatives. Through the development of a novel in vitro approach (PLA: Proximity Ligation Assay), I show that ohmline delocalizes FPN1 from lipid-rafts, thereby decreasing its interaction with its functional partner, ceruloplasmin (CP), a ferroxidase that catalyzes the oxidation of ferrous iron, and consequently its iron export function

A l’heure actuelle, suite à une mauvaise utilisation des antibiotiques, nous faisons face à un problème majeur de santé publique. En effet la résistance aux antibiotiques de certaines souches bactériennes rend le traitement des infections très complexe. Dans ce contexte, le présent projet de thèse concerne l'étude d'un complexe d'efflux bactérien capable de transporter des antibiotiques du cytoplasme vers l'extérieur de la cellule. Ce complexe est composé d'un transporteur de la membrane interne appartenant à la Major Facilitator Superfamily (MFS) (EmrB, E. coli multidrug resistance), d'un canal de la membrane externe TolC (Tolerance to Colicin E1) et d'un adaptateur périplasmique (EmrA, E. coli multidrug resistance). Contrairement aux systèmes d'efflux de type RND (tels que AcrAB-TolC), peu de choses sont connues sur le système EmrAB-TolC de type MFS. Il est donc important d'étudier l'ensemble du complexe sur le plan structurale et fonctionnel afin d'identifier les différences entre ces deux types de systèmes d’efflux. L'objectif de mon projet de thèse était d'étudier au moins un complexe EmrAB-TolC d'un point de vue structurale. Ainsi durant mes études, le but était d'isoler le complexe directement des bactéries surexprimant les trois partenaires protéiques. Dans un premier temps, 15 systèmes homologues EmrAB-TolC ont été identifiés et leurs gènes correspondants amplifiés à partir de l'ADN génomique de différentes bactéries à Gram négatif. Parmi les gènes des 15 systèmes, les gènes codant pour les systèmes d’E. coli et de V. cholerae ont été étudiés plus en détail. Les vecteurs d'expression codaient pour des marqueurs fluorescents pour la mesure des niveaux d'expression de différentes protéines et pour l'étude de la formation des complexes. Dans un premier temps, les différents niveaux d'expression des protéines (EmrB-mRFP1 et EmrA-sfGFP) ont été étudiés pour plusieurs souches d'expression d'E. coli en mesurant les niveaux de fluorescence rouge et verte et par Western blot (anti-His, Myc et Strep pour EmrB, EmrA et TolC). La souche d'E. coli C41(DE3) était la mieux adaptée pour la co-expression d’EmrAB-TolC. Dans un deuxième temps, la méthodologie FSEC (Fluorescence detection Size Exclusion Chromatography) a été utilisée pour identifier un complexe adapté à l'étude structurale. Ainsi, cette méthode a permis d'observer que le complexe EmrAB-TolC d'E. coli était produit en plus grande quantité que celui de V. cholerae. Le protocole final de co-purification consiste à effectuer une lyse douce des bactéries à l'aide du lysozyme, puis après solubilisation avec le DDM, la purification est débutée par une étape de chromatographie d'affinité Ni2+-NTA suivie d'une étape de chromatographie d'exclusion stérique. Enfin, les fractions contenant les trois partenaires protéiques sont utilisées pour l'échange de détergent par l'amphipol A8-35 avant l'étude structurale par microscopie électronique. Les images de microscopie électronique en coloration négative montrent des objets allongés d'une longueur de 33 nm en vue de côté. Une image moyenne d'EmrAB-TolC montre des similitudes avec celle du complexe AcrAB-TolC observé dans des conditions similaires. Les similitudes concernent les densités caractéristiques de TolC. Des différences ont été trouvées pour la partie inférieure d'EmrAB qui est plus fine que la partie inférieure d'AcrAB. Les densités visibles au-dessus de l'anneau d'amphipol correspondent à EmrA, qui présente une structure en forme de canal comme observé avec AcrA. Le canal semble cependant s'étendre plus loin vers la ceinture d'amphipol. Comme EmrB n'a pas de domaine périplasmique étendu présent dans le cas des protéines RND, ces densités sont donc uniquement attribuées à EmrA. EmrA, de l'autre côté, contacte TolC de manière similaire à l'interaction d'AcrA/MexA avec leurs canaux de la membrane externe respectifs (TolC/OprM) de façon «tip-to-tip»
Currently, due to the misuse of antibiotics, we are facing a major public health problem. The resistance to antibiotics of certain bacterial strains makes the treatment of infections very complex. In this context, the present thesis project concerns the study of a bacterial efflux complex capable of transporting antibiotics from the cytoplasm to the outside of the cell. This complex is composed of an inner-membrane Major Facilitator Superfamily (MFS) transporter (EmrB, E. coli multidrug resistance), a channel of the outer membrane TolC (Tolerance to Colicin E1) and a periplasmic adapter (EmrA, E. coli multidrug resistance). Unlike RND-type efflux systems (such as AcrAB-TolC), little is known about the MFS-type EmrAB-TolC system. It is therefore important to study the entire complex on a structural and functional level, to analyse the marked differences between these two types of transport systems. The goal of my thesis project was to study at least one EmrAB-TolC complex from a structural point of view. For my studies the aim was to isolate the complex directly from bacteria overexpressing the three protein partners. In a first step, 15 homologous EmrAB-TolC systems were identified and their corresponding genes amplified from genomic DNA of different Gram-negative bacteria. Among the genes of the 15 systems, the genes coding for the E. coli and V. cholerae systems were further studied. The expression vectors encoded fluorescent markers for the monitoring of the expression levels of different proteins and for studying the formation of complexes. In a first step, the different protein expression levels (EmrB-mRFP1 and EmrA-sfGFP) were studied for several expression strains of E. coli by measuring the red and green fluorescence levels and by Western blot (anti-His, Myc, and Strep for EmrB, EmrA, and TolC). The E. coli strain C41(DE3) was best suited for co-expression of EmrAB-TolC. In a second step, the FSEC (Fluorescence detection Size Exclusion Chromatography) methodology was used to identify a complex suitable for structural study. Thus this method enabled the observation that the EmrAB-TolC complex of E. coli was produced in higher amount than that of V. cholerae. The final co-purification protocol consists in perfoming a gentle lysis of the bacteria using lysozyme, then after solubilization with DDM, the purification is started by a Ni2+-NTA affinity chromatography step followed by a size exclusion chromatography step. Finally, the fractions containing the three protein partners are used for the detergent-exchange by amphipol A8-35 before the structural study by electron microscopy. Negative stain EM-micrographs displayed elongated objects with a length of 33 nm in side view. An average image of EmrAB-TolC shows similarities to that of the AcrAB-TolC complex observed under similar conditions. Similarities included the characteristic densities of TolC. Whereas differences were found in the lower part of EmrAB which is thinner than the lower part of AcrAB. The densities visible above the amphipol-ring correspond to EmrA, which displays a channel-like structure as in AcrA. The channel however seems to extend further towards the amphipol belt. Since EmrB does not have an extended periplasmic domain as the RND proteins have, these densities are therefore solely assigned to EmrA. EmrA, on the other side, contacts TolC akin to the interaction of AcrA/MexA to their cognate outer membrane channels (TolC/OprM) in a ‘tip-to-tip’ fashion
Aufgrund des Missbrauchs von Antibiotika stehen wir derzeit vor einem großen Problem deröffentlichen Gesundheit. Die Antibiotikaresistenz bestimmter Bakterienstämme macht die Behandlungvon Infektionen sehr komplex.In diesem Zusammenhang befasst sich diese Arbeit mit der Untersuchung eines bakteriellenEffluxkomplexes, der Antibiotika vom Zytoplasma zur Außenseite der Zelle transportieren kann. DieserKomplex besteht aus einem Major Facilitator Superfamily (MFS) Transporter der inneren Membran(EmrB, E. coli multidrug resistance), einem Kanal der äußeren Membran TolC (Tolerance to Colicin E1)und einem periplasmatischen Adapter (EmrA, E. coli multidrug resistance).Im Gegensatz zu Effluxsystemen vom RND-Typ (wie AcrAB-TolC) ist über das EmrAB-TolCSystemvom MFS-Typ wenig bekannt. Es ist daher wichtig, den gesamten Komplex auf struktureller undfunktioneller Sicht zu untersuchen, um die deutlichen Unterschiede zwischen diesen beiden Arten vonEffluxsystemen zu analysieren.Ziel meiner Doktorarbeit war es, mindestens einen EmrAB-TolC-Komplex aus struktureller Sichtzu untersuchen. Ziel meiner Studien war es, den Komplex direkt aus Bakterien, die die dreiProteinpartner überexprimieren, zu isolieren. In einem ersten Schritt wurden 15 homologe EmrAB-TolCSystemeidentifiziert und ihre entsprechenden Gene aus der genomischen DNA verschiedenergramnegativer Bakterien amplifiziert. Unter den Genen der 15 Systeme wurden die Gene, die für die E.coli und V. cholerae Systeme kodieren, weiter untersucht. Die Expressionsvektoren codiertenfluoreszierende Marker zur Untersuchung der Expression verschiedener Proteine und zur Untersuchungder Komplexbildung. In einem ersten Schritt wurden die verschiedenen Niveaus der Proteinexpression(EmrB-mRFP1 und EmrA-sfGFP) für mehrere E. coli Expressionsstämme untersucht durch Messen derroten und grünen Fluoreszenzniveaus und durch Western Blot (Anti-His, Myc und Strep für EmrB, EmrAund TolC). Der Stamm von E. coli C41(DE3) war am besten für die Koexpression von EmrAB-TolC14 geeignet. In einem zweiten Schritt wurde die FSEC-Methode (Fluorescence Detection Size ExclusionChromatography) verwendet, um einen für Strukturuntersuchungen geeigneten Komplex zuidentifizieren. Somit konnte mit dieser Methode festgestellt werden, dass der EmrAB-TolC-Komplex vonE. coli in größerer Menge als der von V. cholerae produziert wurde.Das endgültige Ko-Reinigungsprotokoll besteht darin, eine sanfte Lyse der Bakterien unterVerwendung von Lysozym durchzuführen. Nach der Solubilisierung mit DDM wird die Reinigung durcheinen Ni2+-NTA Affinitätschromatographieschritt gefolgt von einemGrößenausschlusschromatographieschritt gestartet. Schließlich werden die Fraktionen, die die dreiProteinpartner enthalten, für den Detergensaustausch durch Amphipol A8-35 vor derStrukturuntersuchung durch Elektronenmikroskopie verwendet.EM-Aufnahmen mit negativer Kontrastierung zeigten längliche Objekte mit einer Länge von 33nm in Seitenansicht. Ein durch Mittlung der Partikel erhaltenes Bild von EmrAB-TolC zeigt Ähnlichkeitenmit dem des AcrAB-TolC-Komplexes, der unter ähnlichen Bedingungen beobachtet wurde.Ähnlichkeiten schlossen die charakteristischen Dichten von TolC ein. Während im unteren Teil vonEmrAB Unterschiede festgestellt wurden, der dünner ist als der untere Teil von AcrAB. Die über demAmphipolring sichtbaren Dichten entsprechen EmrA, das wie bei AcrA eine kanalartige Strukturaufweist. Der Kanal scheint sich jedoch weiter in Richtung des Amphipolgürtels zu erstrecken. Da EmrBkeine erweiterte periplasmatische Domäne aufweist wie die RND-Proteine, werden diese Dichten daherausschließlich EmrA zugeordnet. Auf der anderen Seite kontaktiert EmrA TolC, ähnlich der Interaktionvon AcrA/MexA mit ihren jeweiligen Außenmembrankanälen (TolC/OprM), von “tip-to-tip”

Membrane proteins play an integral role in the control of ion transport across the cell membrane in biological systems. However, due to experimental constraints, structural and functional data available for these proteins is limited, especially considering their importance. In this study, two membrane proteins which transport nitrate and nitrate into the model filamentous ascomycete Aspergillus nidulans were investigated. Work on the twelve trans-membrane domain nitrate transport protein NrtA is well established. As a member of the major facilitator super family (MFS) the role of signature sequences characteristic of this family have previously been studied. Here, a series of point mutations were made to facilitate an understanding of key residues in the nitrate binding domain, the first nitrate signature motif and residues of the unique fungal central-loop domain. Using an expanded alignment package, the proposed secondary structure of NrtA was enhanced and used as a starting point for mutagenesis. Alanine scanning mutagenesis showed that glycine residues in the conserved nitrate nitrite porter (NNP) motif were critical for NrtA function. Two asparagines in the NNP were investigated; N160 and N168. N168 was found to be critical for NrtA function as all mutants were devoid of growth on nitrate solid agar medium though they expressed in the membrane to varying degrees. The nitrate binding site has been studied previously, revealing the interaction of conserved arginine residues with the anion as it traverses the bilayer. Though it was thought that mutations of residue T83 to a small, charge neutral, amino acid would substitute for no alteration to enzyme kinetics in mutant T83S was found when using ¹³NO₃⁻. Another major part of this thesis examined NitA which is part of a distinct nitrite transport family to NrtA (the Formate Nitrite Transporters, FNT). A mutagenesis approach targeted NitA residues conserved amongst homologous proteins. Residues in position D88 in an alignment of homologues were conserved in terms of charge. Mutagenesis of D88 revealed that maintaining charge at this position was essential for NitA function, likely due to a role in salt-bridge formation during conformational changes. Mutations to asparagine, glutamine, serine and valine showed reduced growth on agar though the protein was expressed to approximately wild-type levels. Nitrite uptake assays using a ¹³NO₂⁻ tracer were performed on D88N, D88E and D88Q and all showed wild-type Km and Vmax. Finally, the role of conserved asparagine residues found throughout NitA was investigated by mutagenesis. Expression studies revealed that mutants created in N122 and N246, changed to aspartic acid, lysine, glutamine and serine were generally not present in the membrane and thus did not grow on nitrite agar. However, mutations in N173 (in Tm 4) and N214 (in Tm 5), which are conserved in > 95 % of NitA homologues, showed varying degrees of growth and expression. Both of these residues are located in FNT signature motifs, so it is likely that they are involved with conformational changes or protein dynamics.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Methodological developments and applications of solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy, with particular emphasis on the analysis of protein structure, are described in this thesis. MAS NMR studies of biomolecules ranging from model peptides and proteins in crystalline form to amyloid fibrils and whole bacterial organelles are reported. The methods presented include novel pulse sequences and optimized pulse sequence elements, experimental approaches designed for multiple-spin systems, a protocol for efficient sequential resonance assignment of proteins in the solid state, and techniques to determine the inter-molecular organization of amyloid fibrils formed by moderately sized proteins. Notably, an efficient dipolar recoupling technique, bandselective radio frequency-driven recoupling (BASE RFDR), is introduced and combined with alternating 13C-12C labeling to yield highly sensitive 13C-13C correlation spectra between distant nuclei in proteins. Various applications of the BASE RFDR scheme are presented, including protein resonance assignment, determination of tertiary structure of amyloid fibrils, and variable-temperature studies of protein dynamics. The main biological systems analyzed are amyloid fibrils formed by the SH3 domain of P13 kinase (P13-SH3) and intact gas vesicles from anabaena flos-aquae, for which atomic-level structural information was previously unavailable. P13-SH3 (86 residues) is a system thoroughly studied as a model of protein misfolding and amyloid formation by a natively globular protein. Gas vesicles are bacterial buoyancy organelles, with walls composed almost entirely by a single protein (GvpA, 70 residues), whose formation and structure constitute a highly intriguing biophysical problem. Nearly complete 13C and 'IN resonance assignments and the molecular conformations of the polypeptide backbones of both P13-SH3 and GvpA have been obtained via MAS NMR spectroscopy, enabling the proposal of models for the structure of these two protein assembly systems. In addition, the tertiary structure of P13-SH3 amyloid fibrils has been elucidated by the application of novel methodology introduced in this thesis. Finally, investigations regarding the effects of temperature and protein dynamics on MAS NMR experiments and biomolecular dynamic nuclear polarization studies are presented.
by Marvin J. Bayro.
Ph.D.

DNA repair is essential for survival, as damage to the genome can interrupt the precarious balance of cell functions, causing further mutations and possibly leading to cancer. The bacterial transcription repair coupling factor, Mfd, is capable of recognizing a stalled RNA polymerase at a site of DNA damage. The Mfd works both to remove the RNA polymerase through its motor function (utilizing the energy of ATP to translocate along DNA), and to recruit the DNA repair complex UvrA/B/C. To study conformational changes in the protein, we are creating multiple mutants of the full length Mfd protein. My approach is to use a cleavable mutant of full-length Mfd as a template for further mutations. This will allow us to probe for conformational changes by changing interactions at the interface of the two halves of Mfd, and then using the ability to cut with TEV protease as a sensor to identify and characterize the open state of the protein. By introducing this TEV protease cut site at residue 450 in the protein linker region between the N (amino-) and C (carboxy-) terminal domains, we can then assess the conformational changes Mfd must undergo to obtain activity. We can study the effect of further mutations on the full length and cut versions of the protein. Another approach attempted in this study involves using cysteine modification of the full length Mfd protein as a sensor for these conformational changes. Mfd acts as a model system for studying the DNA repair mechanisms found in humans, and the elucidation of functional and conformational changes in Mfd contributes to studying disease phenotypes resulting from aberrant transcription coupled repair.

The mas proto-oncogene encodes a seven transmembrane protein (MAS) which is suggested to function as a receptor for angiotensin. It (MAS) was initially identified in NIH-3T3 cells that were transformed with DNA isolated from a human epidermoid carcinoma. These cells formed foci in culture and tumors when injected into nude mice. On the other hand, untransformed cells did not. Further analysis of these cells showed that transformed cells bind increased levels of angiotensin when compared to untransformed cells. These studies also demonstrated that the Mas protein was structurally similar to the angiotensin receptor transmembrane proteins, AT1 and AT2 . This investigation was undertaken to examine the ability of the Mas protein to function as an receptor for angiotensin and promote cell proliferation. To this end, quantitation of mas genes by Polymerase Chain Reaction (PCR) and serial dilutions, and Southern blot analysis support an increased in mas genes in transformed cells. Northern blot analysis demonstrated an increased expression of the mas gene in transformed cells. No changes in the level of the AT2 angiotensin receptor gene expression was observed in the transformed and untransformed cell lines. Expression of the AT1 angiotensin receptor gene was not observed in these cell lines. Anti-peptide antibodies were generated against the 1st and 2nd extracellular regions of the Mas protein. Flow cytometric analysis using these antibodies indicated an increased presence of the Mas protein on the surf ace of transformed cells recognized by anti-peptide antibodies. Western blot analysis showed two cross-reacting proteins of approximately ll0kd and 66kd in transformed cells; whereas, only a 66kd protein was found in untransformed cells. Transformed cells exposed to mas antisense oligos greatly reduced the synthesis of Mas, decreased cell proliferation and the binding of angiotensin. Binding studies using [3H]-DUP- 753 (a non-peptidyl ligand which recognizes Ang subtype AT1 receptors) showed little binding to transformed cells. Similar studies using PD-123319 (a non-peptidyl ligand that recognizes AT2 subtype receptors) indicated that approximately 60% of [125I]-Ang II was displaced using PD-123319. Further binding analysis of transformed cells suggests that [Sarl]-Ang II (an Ang II antagonist) could not completely displace [ 125I]-Ang II. Taken together, these data suggest that Mas protein is an Ang receptor which functions in the regulation of cell proliferation. Mas appears to be a member of a subtype different from AT1 or AT2.

Guanidine-stable chymoelastase (GUSCE), a component of the protease mixture known as Pronase, has been shown to be stable and active under conditions which denature and inactivate most proteins. In the absence of denaturant, this enzyme has shown proteolytic specificity for phenylalanyl, tyrosyl, and leucyl peptide bonds. In the presence of denaturant, however, the cleavage specificity has not been defined.In order to determine the effects of denaturant on the cleavage specificity of GUSCE, six small peptides of known amino acid sequence were hydrolyzed by GUSCE in the presence and absence of 6.OM guanidinium chloride. The site of GUSCE cleavage was acertained by dansylation of the new N-terminal amino acids, which were produced by proteolysis, followed by thin layer chromatographic identification of the resulting dansylated amino acids.The results indicate that GUSCE catalyzed the hydrolysis of phenylalanyl and tyrosyl peptide bonds in the absence as well as the presence of 6.OM guanidinium chloride. Of the tyrosyl and phenylalanyl peptide bonds hydrolyzed, all were between non-terminal amino acids, which illistrates the endo-peptidase characteristics of GUSCE. With one exception, only those peptide bonds cleaved by GUSCE in the absence of denaturant were cleaved in the presence of denaturant. In the case of oxytocin, the presence of denaturant was actually required for the cleavage of the Tyr(2)-Ile(3) peptide bond. The demonstrated predictablilty of GUSCE cleavage in the presence of denaturant should greatly enhance its utility in the sitespecific proteolysis of insoluble or otherwise proteolysisresistant protein substrates.Ball State UniversityMuncie, IN 47306

Das Verständnis der Mechanismen, nach denen biologische Systeme ablaufen, ist ein wichtiger Fokus der aktuellen Strukturbiologie. Kernmagnetische Resonanzspektroskopie (NMR) ist eine geeignete Technik, um solche Ziele anzustreben sowie Struktur und Dynamik von Biomolekülen zu erforschen, um komplementäre Informationen zum Verständnis von Proteinfunktionalität zu erhalten. Rasante Fortschritte sind vor nicht langer Zeit auf dem Gebiet biologischer Festkörper-NMR (ssNMR) erzielt worden, was zu vollständiger Strukturaufklärung zahlreicher Peptide und kleiner Proteine, der Beschreibung von Protein-Komplexbildung sowie der der dynamischen Eigenschaften kleiner Proteine geführt hat. Festkörper-NMR ist die Methode der Wahl bei struktureller und dynamischer Charakterisierung von Membranproteinen und aggregierten amyloidogenen Systemen, die schwer löslich und kaum mit Lösungs-NMR oder Röntgenkristallographie zugänglich sind. Moderne Festkörper-NMR ist noch immer limitiert, was Auflösung und Empfindlichkeit betrifft, und macht weitere Entwicklungen auf den Gebieten der Probenpräparation und des Pulssequenz-Designs erforderlich. In meiner Arbeit untersuche ich die potenzielle Verwendung von Deuterierung in der Protein Festkörper-NMR zur Erhöhung von Empfindlichkeit und Auflösung in 15N-1H Korrelationsexperimenten. Der erzielte Fortschritt auf diesen Gebieten erlaubt die Verfolgung von Proteinrückgratbewegungen mit hoher Genauigkeit, die vorher nicht verfügbar war. Wir zeigen zum ersten Mal, dass TROSY Experimente für Festkörper-NMR gewinnbringend sind. Außerdem wurde eine Pulssequenz für 13C-13C J Kopplung zur Erhöhung der Auflösung in der Kohlenstoff-Dimension entwickelt.
Understanding the mechanisms how biological systems work is an important objective of current structural biology. Nuclear magnetic resonance (NMR) spectroscopy is a well suited technique to approach these goals and to study structure and dynamics of biomolecules in order to obtain complimentary information for understanding functionality of proteins. Recently, rapid progress has been made in the field of biological solid state NMR (ssNMR), which resulted in complete structure elucidation of several peptides and small proteins, the characterization of protein complex formation and the characterization of dynamic properties of small proteins. Solid state NMR is the method of choice for structural and dynamic characterization of membrane proteins and aggregated amyloidogenic systems, which are poorly soluble and can not be easily studied by solution state NMR and X-ray spectroscopy. Modern solid state NMR is still limited in resolution and sensitivity, and requires developments in sample preparation and pulse sequence design. In my thesis, I study the potential use of deuteration in protein solid state NMR for sensitivity, as well as for resolution enhancement in 15N-1H correlation experiments. Achieved progress in these fields allows to monitor backbone motion with high accuracy, which has not been available before. We show for the first time that TROSY type experiments can be beneficial for solid state NMR. In addition, a pulse sequence for 13C-13C J decoupling was developed to increase resolution in the carbon dimension.

Mitochondria are important cellular organelles whose functions include generation of ATP, sequestration and release of pro-apoptotic molecules and calcium buffering. Mitochondria function is tightly linked to organelle morphology, which exits in a dynamic spectrum between a highly interconnected/fused mitochondria network to a punctate/fragmented scattering of individual mitochondrion. A family of large GTPase enzymes modulates this spectrum, with fusion catalyzed through the actions of mitofusin 1 and 2 (Mfn1/2) on the outer mitochondria membrane (OMM) and optic atrophy 1 (Opa1) causing fusion of the inner mitochondria membrane (IMM). On the other end of the spectrum, fragmentation is catalyzed through the actions of dynamin-related protein 1 (Drp1). Drp1 is recruited from the cytosol to binding partners at the OMM, organizes into concentric spiral rings, undergoes GTP hydrolysis to constrict the ring and pinches mitochondrion in two. While fragmentation is achieved through the action of only one GTPase enzyme, the mechanisms behind the complex regulation of Drp1 remain relatively obscure. In order to expand upon known Drp1 regulatory mechanisms, an examination of how both Drp1 splicing and Drp1 recruitment to the OMM contributes to protein regulation is necessary. Drp1 contains three alternatively spliced exons, resulting in the potential generation of eight protein isoforms. Each of these isoforms is capable of inducing mitochondrial fragmentation, however one exon arrangement (termed Drp1-x01) can also bind to microtubules within the cell. Characterization of the Drp1-x01 isoform at both the RNA and protein level indicate an important, yet incompletely characterized, role in immune system biology. Drp1 is capable of interacting with several proteins localized at the OMM. Among these, mitochondria fission factor (Mff) has been implicated in the formation of Drp1 spirals and the eventual fragmentation process. Mff contains four alternatively spliced exons as well as several phosphorylation sites identified through nonbiased phosphoproteomic screens. Inclusion of alternative exons to the Mff structure decreases its ability to recruit Drp1 from the cytosol, while phosphomimetic substitutions to conserved serine residues enhances the Drp1::Mff interaction. Taken together, this suggests that regulation of mitochondrial fragmentation occurs at the pretranslational (alternative splicing) and the posttranslational (phosphorylation) level is critical for maintaining the complex, yet essential, balance between mitochondrial fission and fragmentation.

The human brain weighs around 2% of the total body mass, nevertheless it consumes about 20% of the total glucose intake. Glucose, the main energy source of the brain, is important for many processes, for instance as energy for synthesis of neurotransmitters. Therefore a stable glucose concentration in the brain is crucial. Unlike other macronutrients, glucose is able to cross the BBB through facilitated transport by glucose transporters (GLUTs) that belong to the solute carrier (SLC) superfamily. There are currently 65 SLC families with over 400 members in total. Out of 65 families, many belong to the protein family (Pfam) class major facilitator superfamily (MFS). There were 28 putative SLC transporters, 18 of them were called major facilitator domain containing proteins (MFSDs). Recently MFSD2A, MFSD2B, MFSD4A, MFSD4B and MFSD5 were grouped into SLC families making the total amount of current putative SLCs 23 In this project the effects of glucose starvation on MFSD6, MFSD6L, MFSD8, MFSD9 and MFSD10 in primary mouse cortex cultures were studied on protein and gene level through immunocytochemistry (ICC) and quantitative polymerase chain reaction (qPCR). All proteins except for MFSD10 were detected in the ICC. All except MFSD8 displayed a change in fluorescent intensity. MFSD6, MFSD6L and MFSD9 were upregulated after 3 h of glucose starvation compared with control. Gene expression was detected for all targets except for Mfsd6l. Gene expression alterations were found for Mfsd8, Mfsd9 and Mfsd10. The 3 h glucose starvation resulted in an acute response in the gene expression for Mfsd9 and Mfsd10 but was back to control levels after 12 h, while Mfsd8 respond after 12 h of glucose starvation. All of them were back at similar levels as controls when re-fed with glucose. In conclusion, all five MFSDs responded to glucose starvation at some point. For instance MFSD6 responds to glucose starvation on a protein level, Mfsd6 was however also the only gene out of the four tested that did not respond to glucose starvation on a gene level.

Although the basic structure of biological membranes is provided by the lipid bilayer, most of the specific functions are carried out by membrane proteins (MPs) such as channels, ion-pumps and receptors. Additionally, it is known, that mutations in MPs are directly or indirectly involved in many diseases. Thus, structure determination of MPs is of major interest not only in structural biology but also in pharmacology, especially for drug development. Advances in structural biology of membrane proteins (MPs) have been strongly supported by the success of three leading techniques: X-ray crystallography, electron microscopy and solution NMR spectroscopy. However, X-ray crystallography and electron microscopy, require highly diffracting 3D or 2D crystals, respectively. Today, structure determination of non-crystalline solid protein preparations has been made possible through rapid progress of solid-state MAS NMR methodology for biological systems. Castellani et. al. solved and refined the first structure of a microcrystalline protein using only solid-state MAS NMR spectroscopy. These successful application open up perspectives to access systems that are difficult to crystallise or that form large heterogeneous complexes and insoluble aggregates, for example ligands bound to a MP-receptor, protein fibrils and heterogeneous proteins aggregates. Solid-state MAS NMR spectroscopy is in principle well suited to study MP at atomic resolution. In this thesis, different types of MP preparations were tested for their suitability to be studied by solid-state MAS NMR. Proteoliposomes, poorly diffracting 2D crystals and a PEG precipitate of the outer membrane protein G (OmpG) were prepared as a model system for large MPs. Results from this work, combined with data found in the literature, show that highly diffracting crystalline material is not a prerequirement for structural analysis of MPs by solid-state MAS NMR. Instead, it is possible to use non-diffracting 3D crystals, MP precipitates, poorly diffracting 2D crystals and proteoliposomes. For the latter two types of preparations, the MP is reconstituted into a lipid bilayer, which thus allows the structural investigation in a quasi-native environment. In addition, to prepare a MP sample for solid-state MAS NMR it is possible to use screening methods, that are well established for 3D and 2D crystallisation of MPs. Hopefully, these findings will open a fourth method for structural investigation of MP. The prerequisite for structural studies by NMR in general, and the most time consuming step, is always the assignment of resonances to specific nuclei within the protein. Since the last few years an ever-increasing number of assignments from solid-state MAS NMR of uniformly carbon and nitrogen labelled samples is being reported, mostly for small proteins of up to around 150 amino acids in length. However, the complexity of the spectra increases with increasing molecular weight of the protein. Thus the conventional assignment strategies developed for small proteins do not yield a sufficiently high degree of assignment for the large MP OmpG (281 amino acids). Therefore, a new assignment strategy to find starting points for large MPs was devised. The assignment procedure is based on a sample with [2,3-13C, 15N]-labelled Tyr and Phe and uniformly labelled alanine and glycine. This labelling pattern reduces the spectral overlap as well as the number of assignment possibilities. In order to extend the assignment, four other specifically labelled OmpG samples were used. The assignment procedure starts with the identification of the spin systems of each labelled amino acid using 2D 13C-13C and 3D NCACX correlation experiments. In a second step, 2D and 3D NCOCX type experiments are used for the sequential assignment of the observed resonances to specific nuclei in the OmpG amino acid sequence. Additionally, it was shown in this work, that biosynthetically site directed labelled samples, which are normally used to observe long-range correlations, were helpful to confirm the assignment. Another approach to find assignment starting points in large protein systems, is the use of spectroscopic filtering techniques. A filtering block that selects methyl resonances was used to find further assignment starting points for OmpG. Combining all these techniques, it was possible to assign nearly 50 % of the observed signals to the OmpG sequence. Using this information, a prediction of the secondary structure elements of OmpG was possible. Most of the calculated motifs were in good aggreement with the crystal structures of OmpG. The approaches presented here should be applicable to a wide variety of MPs and MP-complexes and should thus open a new avenue for the structural biology of MPs.
Biologische Membranen bestehen hauptsächlich aus Lipiden, ihre Funktion wird jedoch vor allem durch die eingebetteten Membranproteine (z.B. Kanäle, Ionenpumpen und Rezeptoren) bestimmt. Mutationen in dieser Proteinklasse können zum Auftreten verschiedener Krankheitsbilder führen, weshalb die Untersuchung der dreidimensionalen Struktur von Membranproteinen nicht nur von strukturbiologischem, sondern auch von pharmakologischem Interesse ist. In den letzten Jahren wurde eine Methode, die Festkörper NMR Spektroskopie, für Strukturuntersuchungen an Proteinproben im festen Aggregatzustand entwickelt. Diese Arbeit beschäftigt sich mit drei verschiedenen Präparationsarten von Membranproteinen, die eine Aufnahme von hochaufgelösten Festkörper NMR Spektren erlauben. Als Modelsystem wurde das Protein G der äußeren Membrane (outer membrane protein G, OmpG) von Escherichia coli gewählt. Eine wichtige Vorraussetzung zur Berechnung der Proteinstruktur aus den NMR-Spektren, ist die Zuordnung der einzelnen Signale zur jeweiligen Aminosäure in der Proteinsequenz. In dieser Arbeit wurde eine Methode entwickelt, die das Auffinden von Startpunkten für die sequentielle Zuordnung in großen Membranproteinen, wie zum Bsp. OmpG (281 Aminosäuren), erlaubt. Multidimensionale NMR Experimente mit verschieden spezifisch markierten Proben wurden durchgeführt und ermöglichten die Zuordnung von 50 % der NMR Signale der OmpG Proteinsequenz. Zur Überprüfung der gewonnenen Daten wurden diese zur Vorhersage von Sekundärstrukturelementen genutzt. Es konnte gezeigt werden, dass die berechneten Strukturmotive in guter Übereinstimmung zu den bisher veröffentlichten OmpG Strukturen liegen. Die in dieser Arbeit angewendeten Methoden sollten auf eine Vielzahl anderer Membranprotein anwendbar und somit einen neuen Weg zur Strukturbiologischen Untersuchung von Membranproteinen eröffnen.

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors’ function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static 15N NMR spectra and quantitative determination of 1H-13C order parameters through measurement of the 1H-13C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (Sbackbone = 0.59-0.67, SCH2 = 0.41-0.51 and SCH3 = 0.22) obtained in directly polarized 13C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

Les récepteurs couplés aux protéines G de classe A (RCPG) constituent la plus grande famille de récepteurs transmembranaires du génome humain et sont impliqués dans la régulation de nombreux mécanismes physiologiques. Comprendre les mécanismes évolutifs qui ont conduit à la diversité de cette famille de récepteurs pourrait permettre une meilleure connaissance des relations séquence-structure-fonction des différentes sous-familles. Pour obtenir des informations sur l'évolution des RCPG, nous avons exploré leur espace de séquences par multidimensional scaling métrique (MDS). Nous avons appliqué une nouvelle technique MDS qui projette des séquences supplémentaires sur un espace de référence et permet ainsi la comparaison des séquences de différentes espèces. Les résultats montrent que les récepteurs se répartissent en quatre groupes et suggèrent que les récepteurs actuels ont évolué à partir d'ancêtres des récepteurs de peptides suivant trois directions évolutives principales. Les prolines des hélices transmembranaires 2 et/ou 5 sont impliquées dans deux de ces directions. Pour comprendre le mécanisme fin ayant abouti à la formation des différentes sous-familles, nous avons analysé les covariations des résidus à différents niveaux hiérarchiques (classe/groupe/sous-famille). Nous avons testé différentes méthodes pour analyser les mutations corrélées afin de sélectionner une méthode robuste pour les différents jeux de séquences. L'application de cette méthode met en évidence des résidus spécifiques qui sont cruciaux pour l'évolution de sous-familles particulières.

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors’ function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static 15N NMR spectra and quantitative determination of 1H-13C order parameters through measurement of the 1H-13C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (Sbackbone = 0.59-0.67, SCH2 = 0.41-0.51 and SCH3 = 0.22) obtained in directly polarized 13C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

Im Rahmen der vorliegenden Arbeit, wird ein neuartiges Markierungsschema für die Festkörper-NMR-Spektroskopie vorgestellt, das sogenannte Reduced Adjoining Protonation (RAP) Schema, welches die Protonendetektion sämtlicher Aliphaten erlaubt. Hochaufgelöste, 1H-detektierte 1H,13C Korrelationsspektren wurden erhalten. Des Weiteren wurde der Vorteil von hohen MAS-Frequenzen untersucht. 1H- und 13C-detektierte 3D Zuordnungsexperimente wurden implementiert, welche uns die Zuordnung von 90% aller aliphatischen Resonanzen von alpha-Spektrin SH3 erlaubten. Da die chemische Verschiebung abhängt vom Strukturmotiv, kann sie verwendet werden, um Sekundärstruktur-Informationen abzuleiten. Darüber hinaus wurde ein 1H-detektiertes H(H)CH 3D Experiment entwickelt, um weitreichende 1H,1H Kontakte zu ermitteln, welche für die Bestimmung der Tertiärstruktur genutzt werden können. Um artefaktfreie Relaxationsdaten zu erhalten, wurde das RAP-Markierungsschema modifiziert, um 1H- und 13C-verdünnte Proben zu erhalten, in denen Spindiffusion unterdrückt ist. Für die Untersuchung von Sub-Mikrosekunden-Dynamik werden Experimente vorgestellt zur Bestimmung von 13C T1 Relaxationszeiten und 1H,13C dipolaren Kopplungstensoren für Rückgrat- und Seitenketten-Resonanzen. Des weiteren zeigen wir, dass das RAP-Markierungsschema auf nicht-kristalline Systeme, wie Amyloidfibrillen des Abeta1-40 Peptids der Alzheimer-Krankheit, angewendet werden kann. Unter Verwendung von 1H-Detektion, erhielten wir hochaufgelöste 1H,13C Korrelationsspektren. Schließlich wurde der Perdeuterierungsansatz auf den L7Ae-box C/D Protein-RNA Komplex aus P. furiosus angewendet. Wir erhielten hochaufgelöste, 1H-detektierte 1H,15N, sowie 13C,13C Korrelationsspektren des Protein-RNA Komplexes. Weiterhin haben wir eine Methode zur Bestimmung genauer Abstands- und Winkelinformationen für die Protein-RNA Schnittstelle etabliert und schlagen Ansätze vor, für die Zuordnung der chemischen Verschiebungen von RNA-Resonanzen.
In this thesis, a novel labeling scheme for solid-state NMR spectroscopy, the Reduced Adjoining Protonation (RAP) scheme, is introduced, which allows proton detection of all aliphatic sites, as shown for the microcrystalline SH3 domain of alpha-spectrin. These samples yield high-resolution, 1H-detected 1H,13C correlation spectra. In addition, the benefit of high MAS frequencies was investigated. 1H- and 13C-detected 3D assignment experiments are implemented, which allowed us to assign 90% of all aliphatic resonances of alpha-spectrin SH3. As the chemical shift is dependent on the structural motif, it can be employed to derive secondary structure information. Furthermore, a 1H-detected H(H)CH 3D experiment is introduced, to obtain long-range 1H,1H contacts, which can be used for the determination of the tertiary structure. To obtain artifact-free relaxation data, the RAP labeling scheme was modified to obtain sparsely proton labeled, 13C dilute samples, in which spin diffusion is suppressed. To probe sub-microsecond dynamics, we report experiments to determine 13C T1 relaxation times and 1H,13C dipolar coupling tensors for backbone and side chain resonances, respectively. Furthermore, we show, that the RAP labeling scheme can be applied to non-crystalline systems, such as amyloid fibrils of the Alzheimer’s disease peptide Abeta1-40. Using 1H-detection, we obtained high-resolution 1H,13C correlation spectra. Finally, we applied the perdeuteration approach to the L7Ae-box C/D protein-RNA complex from P. furiosus. We obtained high-resolution, 1H-detected 1H,15N, as well as 13C,13C correlation spectra of the protein-RNA complex. In addition, we established a methodology to determine accurate distance and angular restraints for the protein-RNA interface and propose approaches for the chemical shift assignment of RNA resonances.

Abstract The mast cells are an integral part of the human immune system. They are important modulators of inflammatory and physiological processes. Mast cells exert their functions through degranulation and release of inflammatory mediators, such as histamine, proteases and cytokines. There are two main pathways leading to the mast cell activation, the immunoglobulin-dependent and the immunoglobulin-independent pathway. The latter pathway can be triggered by several non-immunological stimuli, and two novel receptors responsible for the activation have been identified, the MAS-related G protein-coupled receptor X1 (MRGPRX1) and X2. The MRGPRX1 and MRGPRX2 have two established functions: i) they trigger the degranulation of mast cells and ii) they are involved in pain perception and itch on a specific subset of sensory neurons. These receptors are not expressed in all of the populations of mast cells, only in the tryptase and chymase containing mast cells, contributing to the mast cell heterogeneity. Unlike most G protein-coupled receptors, the MRGPRX1 and MRGPRX2 are quite non-selective, binding an ever growing list of different ligands. Their ligands include endogenous neuropeptides, host defense peptides and protein fragments, as well as synthetic compounds such as different antibiotics. Their endogenous ligands could be a triggering signal in some mast cell-related diseases by degranulating mast cells and thereby inducing inflammation. Due to the non-selectivity of MRGPRX1 and MRGPRX2, they probably still have many hitherto unknown ligands. The aim of this study was to isolate novel endogenous ligands for the MRGPRX1 and MRGPRX2 from human tissues with the “reverse pharmacology approach” and to determine their potential to degranulate mast cells. The starting materials for the isolation, human platelets and plasma, contained MRGPRX1 and MRGPRX2 activating compounds. From the human plasma, three fragments of albumin able to activate the MRGPRX2 were isolated and sequenced. These fragments were dose-dependently activating the MRGPRX2 and degranulating mast cells. Two MRGPRX1 activating hemoglobin β-chain fragments were isolated from human platelets. These fragments were dose-dependently activating the MRGPRX1, but had no effect on mast cell degranulation
Tiivistelmä Syöttösolut on tärkeä osa ihmisen immuunijärjestelmää. Ne ovat tärkeitä tulehdus- ja fysiologistenprosessien säätelijöitä. Syöttösolujen vaikutus välittyy degranulaation ja siinä vapautuvien tulehdusvälittäjäaineiden kautta. Vapautuviin aineisiin lukeutuu esim. histamiini ja lukuisia sytokiinejä, sekä proteaaseja. Syöttösolujen aktivaatio voi tapahtua immunoglobuliineista riippuvaa tai immunoglobuliineista riippumatonta reittiä pitkin. Monet ei-immunologiset tekijät voivat laukaista jälkimmäisen reitin ja kaksi uutta tähän vaikuttavaa G-proteiinikytkentäistä reseptoria on löydetty, MAS-related G protein-coupled receptor X1 (MRGPRX1) ja X2. MRGPRX1:llä ja MRGPRX2:lla on kaksi tunnettua tehtävää: i) ne laukaisevat syöttösolujen degranulaation ja ii) ne osallistuvat kivun ja kutinan aistimiseen tietyissä tuntohermoissa. Näitä reseptoreita ei ilmennetä kaikissa syöttösoluissa, vaan ainoastaa tryptaasia ja kymaasia sisältävissä syöttösoluissa, ja täten osaltaan selittävät syöttösolujen monimuotoisuutta. Useimmista G-proteiinikytkentäisistä reseptoreista poiketen MRGPRX1 ja MRGPRX2 ovat laajakirjoisia, sitoen monia erilaisia ligandeja. Ligandeihin kuuluu endogeenisia neuropeptidejä, antimikrobiaalisia peptidejä ja proteiinin fragmentteja, sekä synteettisiä yhdisteitä kuten erilaisia antibiootteja. Reseptoreiden endogeeniset ligandit voivat toimia laukaisijana jossain syöttösoluihin liittyvissä sairauksissa, degranuloidessaan syöttösoluja ja aiheuttaen paikallisen tulehdustilan. Reseptoreiden laajakirjoisuudesta johtuen niillä on oletettavasti monia vielä tuntemattomia ligandeja. Tämän tutkimuksen tarkoitus oli eristää uusia endogeenisiä ligandeja MRGPRX1:lle ja MRGPRX2:lle ihmisen kudoksista ”kääteisfarmakologista lähestymistapaa” hyödyntäen ja selvittää ligandien kyky syöttösolujen degranulaatioon. Lähtömateriaalina käytetyt ihmisen verihiutaleet ja plasma sisälsivät MRGPRX1:ta ja MRGPRX2:ta aktivoivia yhdisteitä. Plasmasta eristettiin ja sekvensoitiin kolme albumiinin fragmenttia, jotka aktivoivat MRGPRX2:ta. Nämä fragmentit aktivoivat MRGPRX2:ta ja degranuloivat syöttösoluja annosriippuvaisesti. Kaksi MRGPRX1:tä aktivoivaa hemoglobiinin β-ketjun fragmenttia eristettiin ihmisen verihiutaleista. Nämä fragmentit tunnistettiin hemorfiineiksi ja ne aktivoivat MRGPRX1:tä annosriippuvaisesti, mutta eivät vaikuttaneet syöttösolujen degranulaatioon

The multidrug transporter LmrP, member of the major facilitator superfamily (MFS), confers L. lactis and recombinant E. coli cells resistance to an array of cytotoxic compounds including antibiotics. LmrP mediates drug extrusion from the plasma membrane by an electrogenic proton/drug exchange reaction, whereby a positively charged substrate may move towards the external medium in exchange for two or more protons moving towards the cytoplasm. Recent studies have suggested that MFS transporters require phosphatidylethanolamine (PE) for function and proper topology. However, the specificity of the PE requirement, as well as the contribution of the electrochemical gradient (the driving force of the substrate transport) to this lipid requirement was not addressed. Here we report a new approach for addressing PE specific requirement for the function and the structure of membranes transporters. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by phosphatidylcholine (PC) ( including energy transfer between the protein tryptophan residues and the lipid headgroups). We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Some yeast strains under certain conditions secrete into the medium polypeptide toxins which are inhibitory to sensitive cells. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component &
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-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. We have previously characterized the K5 type killer protein. This protein is an exo &
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-1,3-glucanase which is stable at pH&rsquo
s and temperatures appropriate for its medical usage. &
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-1,3- glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antimycotic agent. Antifungal activity of the K5 type yeast killer protein was tested against 26 human pathogenic yeast and 9 dermathophyte strains and found to be affective on all of the tested strains. Toxin MIC50, MIC100 and MFC values were found to be between 0.25-4, 0.5-8, 1-8 µ
g/ml respectively except Candida krusei isolates. Cell killing analysis revealed that toxin activity starts within first 2 hours and complete cell death time differs due to the susceptibility of strains to the K5 type yeast killer protein. K5 type yeast killer protein would be used as a novel and selective agents with the results obtained from this study.

À ce jour, nos connaissances sur les propriétés structurales et fonctionnelles des métalloprotéines sont essentiellement basées sur des structures résolues par des méthodes de diffraction à rayons X appliquées à des échantillons monocristallins. Cependant, certaines protéines ne cristallisent pas ou cristallisent sous une forme qui n’est pas manipulable ou compatible avec des techniques des diffraction, et même si une structure à très haute résolution est disponible, la nature de l’ion métallique, sa géométrie de coordination ou son état d’oxydation restent souvent indéterminés.La Résonance Magnétique Nucléaire en rotation à l’angle magique (MAS NMR) est une technique très performante pour l’étude de systèmes biologiques et pour la caractérisation de la structure du site actif des métalloprotéines paramagnétiques, mais son application à l’analyse des noyaux proches d’un site paramagnétique est limitée à cause de la résolution et de la sensibilité faibles.L’objectif de cette thèse a été de développer des méthodes RMN basées sur des hautes fréquences de rotation (60-111 kHz MAS) pour faire face à ces problématiques. Un répertoire de séquences d’impulsion pour la détection et l’attribution des noyaux à proximité d’un centre paramagnétique est proposé, et à l’aide de méthodes de calculs de pointes, les données expérimentales acquises sont converties en contraintes structurales afin de déterminer la géométrie du site actif à l’échelle atomique. Cette approche est validée avec l’analyse de sites actifs de deux protéines microcristallines contenants différents ions paramagnétiques : Fe, Cu et Co. Ensuite, des données préliminaires sur un transporteur membranaire d’ions métalliques divalents non cristalline sont présentées.Les méthodes analytiques présentées ici constituent un ensemble d’outils indispensable pour l’élucidation de la structure et la fonction des sites métalliques de systèmes macromoléculaires biologiques
Most of our understanding of metalloproteins derives from atomic or molecular structures obtained from diffraction methods on single crystal samples. However, not all proteins are amenable for diffraction studies, and even when a highly-resolved structure is available, often the nature of the metal ion, its coordination geometry or its oxidation state are not determined. The aim of the present thesis is the investigation of structural properties of metal sites in paramagnetic metalloproteins by Magic-Angle Spinning Nuclear Magnetic Resonance (MAS NMR). MAS NMR is a powerful technique for the investigation of biological systems, and may represent a direct probe of the structure at the active site of paramagnetic metalloproteins. However, it suffers from limited sensitivity and resolution when applied to nuclei close to a paramagnetic center.In this thesis, we address these limitations by developing NMR methods based on ultra-fast (60-111 kHz) MAS rates. A “toolkit” of suitably designed pulse sequences is built for the detection and the assignment of nuclei in close proximity of a paramagnetic center. State-of-the-art computational techniques are also employed to convert the experimental data into structural restraints for obtaining atomic-resolution geometries of active sites. We benchmark this approach with the study of Fe, Cu and Co sites in two microcrystalline proteins, and we also provide preliminary data on a non-diffracting divalent metal ion transporter in lipid membranes. We anticipate that the techniques described here are an essential tool to elucidate many currently unanswered questions about structure and function of metal sites in structural biology

High-resolution solid-state NMR (ssNMR) has recently emerged as a powerful characterization technique for systems that cannot be investigated by solution NMR or X-ray crystallographic methods, and represents a subtle complementary technique for any atomic-scaled study. This is particularly true in structural biology. There exist nowadays well established protocols for sample preparation, resonance assignment and collection of structural restraints, that have paved the way to the first three-dimensional structure determinations at atomic resolution of biomolecules in the solid state, from microcrystalline samples to fibrils and membrane-associated systems. Despite rapid uptake in the field of structural biology, however, these methods for structure determination are far from being routine, and several important problems remain however to be solved before ssNMR is applied to the study of challenging solid protein assemblies. Many methodological developments are still expected in this fast evolving field. Most of the model systems used up-to-date for method development in biological solid-state NMR, are relatively small globular proteins, in the range of 50 to 80 residues (approximately 5.5 to 9.5 kDa). In order to extend the capabilities of ssNMR to larger substrates, the objectives of this thesis are twofold: a) to establish a new, large and more complex model system, and b) to develop new, sophisticated NMR experiments in order to improve the sensitivity and the resolution of the currently existing schemes for resonance assignment, which is one of the main barrier to progress to structural investigation in solid proteins. The N-terminal domain of the subunit of E. coli DNA polymerase III (186 : 186 residues, 18 kDa) was selected as a target. This domain represents the catalytic core of the E. coli replisome, the large molecular machine that replicated DNA in bacteria. In a first part, preparation conditions for solid-state NMR are obtained, notably in combination with automated screening processes for high-throughput protein crystallography, and almost complete resonance assignment is performed by the application of established experiments based on high-power rf irradiations and slow magic-angle spinning (MAS) at high magnetic fields. In a second part, we explore the use of MAS at so-called ultra-fast spinning rates (60 kHz). We show that this makes possible the use of “totally low power” experiments. This yields an extraordinary increase in resolution and sensitivity, enabling the acquisition of selective cross- polarization (CP) transfers, through-bond correlations and 1 H-detected correlations. In particular, we demonstrate that narrow 1 H NMR line widths can be obtained for fully protonated protein samples in the solid state under ultra-fast magic-angle spinning for medium-size microcrystalline and non-crystalline proteins, without any need for dilution against a deuterated background. This provides extensive, robust and expeditious assignments of the backbone 1 H, 15 N, 13 Cα and 13 CO resonances of proteins in different aggregation states, without the need of deuteration. The final part of this thesis concerns the study of thermotropic liquid crystals (LC or LX) phases of a de Vries smectogen, the (S)-hexyl-lactate derivative abbreviated as 9HL, selectively deuterated in a phenyl moiety of the aromatic core. de Vries mesophases show a substantially constant layer spacing in the transition between smectic C and smectic A mesophases and are for this reason of great interest for the development of new ferroelectric (FLC) and antiferroelectric (AFLC) electrooptic devices. Our work is the first attempt to apply NMR to characterize the nature of the de Vries transition, discriminating among possible models. It is also one of the first examples in the scientific literature of application of high magnetic field (above 16 T) for the analysis of LX phases.

Control and analysis of protein aggregation is an increasing challenge to biopharmaceutical research and development. Therefore it is important to understand the interactions, causes and analysis of particles in order to control protein aggregation to enable successful biopharmaceutical formulations. This work investigates the role of different non-ionic surfactants on protein conformational stability, as assessed by HSDSC, and on protein size stability as assessed by Dynamic Light Scattering (DLS), HIAC and MFI. BSA and IgG2 were used as model proteins. Thermal unfolding experiments indicated a very weak surfactant-immunoglobulin IgG2 interaction, compared to much stronger interactions for the BSA surfactant systems. The DLS results showed that BSA and IgG2 with different surfactants and concentration produced different levels of particle size growth. The heat treatment and aging of samples in the presence of Tween 20, Tween 80, Brij 35 and Pluronic F-68 surfactants led to an increase in the populations of larger particles for BSA samples, whereas IgG2 systems did not notably aggregate under storage conditions MFI was shown to be more sensitive than HIAC technique for measuring sub-visible particles in protein surfactant systems. Heat treatment and storage stress showed a significant effect on BSA and IgG2 protein sub-visible particle size stability. This work has demonstrated that both proteins with different Tween 20, Tween 80, Brij 35 and Pluronic F-68 concentrations, have different level of conformational and size stability. Also aging samples and heating stress bears the potential to generate particles, but this depends on surfactant type. Poor predictive correlations between the analytical methods were determined.

L’apoptosi e il ciclo cellulare privi di normali controlli sono i principali motivi che determinano la mancata risposta dell’LLC nei confronti della chemioterapia convenzionale. Nella LLC-B sembra che il ciclo cellulare sia arrestato alla fase G0/G1, l’inibizione della spontanea apoptosi e l’up-regolazione dell’espressione della proteina anti-apoptotica bcl-2 definisce la prognosi clinica. Comunque, esistono delle evidenze che la progressione della malattia è correlate con il ciclo cellulare delle cellule nella LLC-B: un pool di cellule proliferanti è stato riscontrato nei linfonodi e nel midollo osseo e potrebbe rifornire il pool di cellule in accumulo nel sangue periferico. Inoltre, la presenza di mutazioni nel gene che codifica per le catene leggere delle immunoglobuline (Ig) VH si correla con una rapida progressione di malattia (DP) ed una ridotta sopravvivenza (OS) (Damle, Hamblin, 1999). Le cellule B di pazienti affetti da LLC che presentano geni IgVH non mutati esprimono l’RNA ZAP 70 che codifica per la proteina ZAP-70, una proteina tirosin chinasi di 70-kDa, che ha proprietà di molecola segnale, associata ad un incremento delle cellule B e ad un aumento del rischio di progressione della malattia nella LLC-B. (Del Principe, 2006). Inoltre, ad oggi la disponibilità di rinfamicina o inibitori dei proteosomi contro la proliferazione di cellule B nella LLC e l’uso di oligonucleotidi antisenso bcl-2 ci ha suggerito di valutare il reale impatto del meccanismo dell’apoptosi e della proliferazione sulla prognosi della LLC-B. I principali scopi del nostro studio sono stati: 1) determinare la sopravvivenza libera da progressione (PFS) rispetto all’apoptosi/proliferazione e all’espressione di ZAP-70; 2) se l’apoptosi/proliferazione potrebbe predire percorsi alternativi all’interno del gruppo che presenta ZAP-70; e infine 3) se ZAP-70 e il gruppo apoptosi/proliferazione erano indipendenti da fattori prognostici. Perciò noi abbiamo analizzato 265 pazienti, età mediana di 64 anni (range 37-84), 136 maschi e 129 femmine. In riferimento alla stadio Rai modificato, 87 pazienti avevano un basso stadio, 170 uno stadio intermedio e 8 un alto stadio. ZAP-70 fu quantificato mediante citometria a flusso multicolour utilizzando il metodo di fissaggio e permeabilizzazione ritenendo significativo un valore superiore al 20%. Anche Bcl-2 fu determinata attraverso la citometria a flusso, dividendo per la media dell’intensità di fluorescenza (MFI) delle cellule CD19+B-CLL / MFI delle cellule T (Bcl-2B/T). La threshold fu settata ad un valore mediano >1.6. Il recettore per la Transferrina (CD71) fu utilizzato come stima della proliferazione e la threshold fu settata ad un valore mediano >8%. Combinando Bcl-2B/T con CD71 (Bcl2CD71) sono stati ottenuti tre sottogruppi: 1) Bcl2CD71- [106 pts] con basso livello di proliferazione (CD71 <8%) e alta apoptosi (Bcl-2B/T <1.6); 2) Bcl2CD71+ [49 pts] con alta proliferazione (CD71>8%) e bassa apoptosi (Bcl-2B/T >1.6); e 3) Bcl2CD71+/- [110 pts] con bassa proliferazione e bassa apoptosi oppure con alta proliferazione e alta apoptosi. I pazienti affetti da LLC-B ZAP-70+ erano 95/265 (36%). In 111 pazienti studiati l’espressione di ZAP-70 e lo stato mutazionale del gene Ig V gene erano significativamente correlati (p<0.00001). Inoltre, noi trovammo una significativa associazione sia tra bassi livelli di ZAP-70 e basso Bcl-2B/T index (p=0.001) o basso livello di ZAP-70 e Bcl2CD71- (p=0.002), confermando che bassi livelli di ZAP-70 erano caratterizzati da elevata apoptosi e bassa proliferazione. In riferimento al decorso clinico, una significativa più corta sopravvivenza libera da progressione (PFS) fu osservata nei pazienti ZAP-70+ vs pazienti ZAP-70 negative (0% vs 58% a 13 anni; p<0.00001) e in pazienti Bcl2CD71+ vs pazienti Bcl2CD71- (10% vs 56% a 12 anni; p<0.00001). Il sottogruppo Bcl2CD71+/- mostrava un decorso intermedio (30% a 12 anni). Per analizzare ulteriormente l’impatto prognostico dell’indice Bcl2CD71, noi valutammo la sua espressione all’interno dei pazienti ZAP70+ (95 pts) e ZAP70- (170 pts). In realtà, Bcl2CD71 non fu in grado di identificare un livello prognostico all’interno dei pazienti ZAP-70+, perchè tutti questi pazienti presentavano una più corta PFS senza significative differenze. Del resto, questo indice identifica un livello differente di PFS all’interno del sottogruppo ZAP-70 negativo (73% dei pazienti per Bcl2CD71- vs 29% dei pazienti per Bcl2CD71+ a 12 anni, p=0.00009). Operando una analisi multivariata della PFS, nella quale l’età, lo stadio di Rai modificato, CD38, CD23 solubile (sCD23), raddoppiamento dei linfociti (LDT), Bcl-2CD71 e ZAP-70, ZAP-70 (p=0.00005), LDT (p=0.006), stadio Rai modificato (p=0.03) e sCD23 (p=0.01) risultarono essere dei fattori prognostici indipendenti. Inoltre, si confermò che ZAP-70 era il principale fattore prognostico indipendente con riferimento alla PFS. Del resto, il nostro indice apoptosi/proliferazione (Bcl2CD71), eseguito utilizzando la citometria a flusso, fu molto utilizzato per identificare pazienti a differenti livelli di progressione all’interno del sottogruppo ZAP-70 negativo. Dal momento che ZAP-70 negativo rappresenta un ampio ed eterogeneo gruppo della popolazione di LLC-B con una progressione variabile, altri fattori biologici, come il livello di apoptosi e di proliferazione, devono essere considerati in modo che insieme possano identificare più facilmente i pazienti in progressione e dare modo di prendere in tempo una accurata decisione terapeutica.
Dysfunctional apoptosis and cell cycle are the main reasons for the clinical enigma, that CLL can not yet be cured with conventional chemotherapy. In B-CLL, malignant cells seem to be arrested in the G0/early G1 phase of the cell cycle, and inhibition of spontaneous apoptosis and upregulation of the anti-apoptotic protein bcl-2 define clinical prognosis. However, increasing evidence exists that disease progression relies upon cycling B-CLL cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Moreover, the lack of immunoglobulin (Ig) VH gene mutation also has been shown to predict a rapid disease progression (DP) and an inferior overall survival (OS) (Damle, Hamblin, 1999). B-CLL cells that use non-mutated IgVH genes express ZAP-70 RNA, which encodes ZAP-70, a 70-kDa protein tyrosine kinase, associated both with an enhanced B cell receptor signaling and with an early DP risk in B-CLL (Del Principe, 2006). Moreover, the today availability of rapamycin or proteasome inhibitors effective against proliferating B-CLL cells and bcl-2 antisense oligonucleotides prompted us to evaluate the real impact of proliferation and apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) upon apoptosis/proliferation subgroups and ZAP-70 expression; 2) whether apoptosis/proliferation could predict varied outcome within ZAP-70 subgroups; and finally 3) whether ZAP-70 and apoptosis/proliferation groups were independent prognostic factors. Therefore we investigated 265 pts, median age 64 years (range 37-84), 136 males and 129 females. With regard to modified Rai stages, 87 patients had a low stage, 170 an intermediate stage and 8 a high stage. ZAP-70 was quantified by a multicolor flow cytometric method fixing a cut-off value of 20%. Bcl-2 was determined by flow cytometry, dividing mean fluorescence intensity (MFI) of CD19+B-CLL cells / MFI of T-cells (Bcl-2B/T). The threshold was set at the median value >1.6. Transferrin receptor (CD71) was used as a measure of the proliferation and the threshold was set at the median value >8%. Combining Bcl-2B/T with CD71 (Bcl2CD71) we enucleated three subgroups: 1) Bcl2CD71- [106 pts] with low proliferation (CD71 <8%) and high apoptosis (Bcl-2B/T <1.6); 2) Bcl2CD71+ [49 pts] with high proliferation (CD71>8%) and low apoptosis (Bcl-2B/T >1.6); and 3) Bcl2CD71+/- [110 pts] with low proliferation and low apoptosis or with high proliferation and high apoptosis. ZAP-70+ B-CLL patients were 95/265 (36%). In 111 studied pts ZAP-70 expression and Ig V gene mutational status were significantly correlated (p<0.00001). Furthermore, we found significant associations either between lower ZAP-70 and lower Bcl-2B/T index (p=0.001) or lower ZAP-70 and Bcl2CD71- (p=0.002), confirming that low levels of ZAP-70 were characterized by high apoptosis and low proliferation. With regard to clinical outcome, a significant shorter progression-free survival (PFS) was observed in ZAP-70+ pts vs ZAP-70 negative pts (0% vs 58% at 13 years; p<0.00001) and in Bcl2CD71+ pts vs Bcl2CD71- pts (10% vs 56% at 12 years; p<0.00001). The Bcl2CD71+/- subgroup showed an intermediate outcome (30% at 12 years). To further explore the prognostic impact of Bcl2CD71 index, we investigated its expression within ZAP70+ (95 pts) and ZAP70- (170 pts) subsets. As a matter of fact, Bcl2CD71 was not able to identify prognostic subsets within ZAP-70+ pts, because all these cases presented a shorter PFS without significant differences. On the other hand, this index identified subsets at different PFS within the ZAP-70 negative subgroup (73% for Bcl2CD71- pts vs 29% for Bcl2CD71+ at 12 years, p=0.00009). In multivariate analysis of PFS, in which age, Rai modified stages, CD38, soluble CD23 (sCD23), lymphocyte doubling time (LDT), Bcl-2CD71 and ZAP-70 entered, ZAP-70 (p=0.00005), LDT (p=0.006), Rai modified stages (p=0.03) and sCD23 (p=0.01) resulted to be independent prognostic factors. Therefore, ZAP-70 was confirmed as the most important indipendent prognostic factor with regard to PFS. However, our apoptotic/proliferative index (Bcl2CD71), performed by flow cytometry, was very useful to identify pts at different progression rate within the ZAP-70 negative subgroup. Since the ZAP-70 negative subset represents a large and heterogeneous B-CLL population with a variable progression, other biological factors, such as the amount of apoptosis and the proliferative rate, have to be added in order both to identify early progressive pts and to take timely accurate therapeutic decisions.

Groat oil content and composition are important quality traits in oats (Avena sativa L). These traits are controlled by many genes with additive effects. The chromosomal regions containing these genes, known as quantitative trait loci (QTL), can be discovered through their close association with markers. This study investigated total oil content and fatty acid components in an oat breeding population derived from a cross between high oil ('Dal') and low oil ('Exeter') parents. A genetic map consisting of 475 DArT (Diversity Array Technology) markers spanning 1271.8 cM across 40 linkage groups was constructed. QTL analysis for groat oil content and composition was conducted using grain samples grown at Aberdeen, ID in 1997. QTL analysis for multiple agronomic traits was also conducted using data collected from hill plots and field plots in Ottawa, ON in 2010. QTLs for oil content, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) were identified. Two of the QTLs associated with oil content were also associated with all of the fatty acids examined in this study, and most oil-related QTL showed similar patterns of effect on the fatty acid profile. These results suggest the presence of pleiotropic effects on oil-related traits through influences at specific nodes of the oil synthesis pathway. In addition, 12 QTL-associated markers (likely representing nine unique regions) were associated with plant height, heading date, lodging, and protein content. The results of this study will provide information for molecular breeding as well as insight into the genetic mechanisms controlling oil biosynthesis in oat.

L’allergie de contact est une pathologie de la peau particulièrement répandue dans les pays industrialisés. Aucune thérapie ne permet actuellement de la soigner et seule l’éviction de l’allergène permet de la prévenir. Historiquement, l’évaluation du potentiel sensibilisant des molécules mises sur le marché a toujours été réalisée au moyen de tests sur l’animal. Cependant, le champ d’action de ces tests est aujourd’hui limité en raison de la nouvelle législation européenne sur les cosmétiques. Dans ce contexte, le développement de méthodes alternatives ne reposant pas sur l’utilisation d’animaux devient capital. L’allergie de contact repose sur une étape chimique clé : la formation d’un complexe antigénique allergène-protéine capable d’activer le système immunitaire cutané. Le but de ce travail de thèse a été d’étudier le comportement in situ d’allergènes au sein d’épidermes reconstruits de type SkinEthic®. A l’aide d’une technique d’analyse non invasive, la spectroscopie RMN HRMAS, il a été possible de suivre le devenir de différents allergènes, de leur éventuelle activation par voie métabolique, jusqu’à leur fixation sur les protéines épidermiques
Contact dermatitis is a skin pathology particularly prevalent in industrialized countries. No therapy currently exists and only complete avoidance of the particular allergen can prevent an allergic reaction. Historically, the assessment of skin sensitisation potential of molecules placed on the market was always carried out by animal testing. However, the scope of this testing method is now limited by the new European cosmetics legislation. In this way, the development of alternative methods, not based on animal experimentation, become an important issue. Contact dermatitis results of a chemical key step: the formation of an antigenic complex allergen-protein complexe able to activate the cutaneous immune system. The aim of this PhD work was to study the in situ behaviour of allergens in reconstructed human epidermis (SkinEthic® model). By using an appropriate non-invasive analysis technique, HR-MAS NMR spectroscopy, it has been possible to study the mode of action of different allergens, from their possible activation through the metabolic pathway to the binding with epidermal proteins

Les mécanismes de type prion sont désormais reconnus comme sous-tendant la plupart des maladies neurodégénératives humaines, avec en premier lieu la maladie d’Alzheimer (MA) au niveau de ses 2 marqueurs spécifiques, l’amyloïde β (Aβ à l’origine de l’hypothèse étiopathogénique de la cascade amyloïde) et la protéine Tau phosphorylée. Par ailleurs la protéine du prion (PrPC) est décrite comme interagissant à de multiples niveaux avec le métabolisme de l’Aβ sans que les mécanismes physiopathologiques sous-jacents n’aient pu être expliqués. Pour sortir de l’impasse actuelle concernant le développement d’approches thérapeutiques efficaces pour la MA, l’industrie pharmaceutique a besoin de modèles expérimentaux innovants. En effet, à ce jour aucun modèle in vivo, en dépit des progrès réalisés avec les souris transgéniques, n’arrive à refléter la complexité cérébrale humaine ni à mimer une MA clinique. Les cultures in vitro en 2D sont quant à elles très éloignées des situations conduisant à l’accumulation d’agrégats protéiques pathologiques. Le but de notre thèse a été d’utiliser dans le domaine des neurosciences les nouvelles perspectives de recherche ouvertes par les technologies des cellules souches pluripotentes induites (cellules iPS) en développant un modèle de différentiation en 3D pour obtenir des organoïdes cérébraux humains (OC) (mini cerveaux). Leur capacité d’auto-organisation en 3D de tissu neuroectodermique nous a permis de recréer un système complexe mimant différentes structures cérébrales humaines dans lesquelles nous avons pu caractériser les marqueurs attendus. L’étude de l’expression des protéines d’intérêt APP et PrPC pendant la différentiation neurale a permis de caractériser la modulation des niveaux des deux protéines en fonction du temps de culture. Afin d’orienter le modèle vers des mécanismes d’accumulation protéique de type MA, nous avons testé différents inducteurs chimiques dont l’Aftin-5 qui est capable de moduler les voies post-traductionnelles de l’APP. Plusieurs stratégies de traitement ont été adoptées pour induire le clivage de l’APP et la génération d’Aβ. La production des fragments solubles Aβ38, Aβ40, Aβ42 a été mise en évidence par ELISA. Les niveaux générés sont reproductibles et l’augmentation du ratio Aβ42/Aβ40 est cohérente avec les données extrapolées des modèles murins et humains, ce qui a permis de valider notre modèle. Les niveaux d’expression génique et protéique de PrPC et de APP suite au traitement ont été analysés afin de mieux déterminer le rôle de l’interaction entre ces deux facteurs. L’objectif à long terme consiste à améliorer ce modèle, dont les limites actuelles sont notamment l’absence de vascularisation et le niveau de maturation du tissu neural. Le défi majeur dans le cadre de la culture des OC consiste donc à favoriser l’intégration du système vasculaire, et par ailleurs à accélérer le vieillissement in vitro pour l’étude de maladies neurodégénératives. La perspective de pouvoir automatiser le système de culture des OC permet d’envisager l’utilisation de ce modèle à plus grande échelle dans le cadre de test de cytotoxicité et/ou de criblage pharmacologique à haut débit pour identifier de nouvelles molécules thérapeutiques pour la MA
Prion-like mechanisms are known to underlie most of human neurodegenerative diseases including Alzheimer’s disease (AD), which is characterized by two important pathological markers, β amyloid (or Aβ at the origin of the etiopathogenic amyloid cascade hypothesis) and phosphorylated tau protein. Furthermore, the prion protein (PrPC) interacts at multiple levels with the metabolism of Aβ, by mechanisms which are not well understood. To overcome the current limits in the development of efficient strategies to treat AD, the pharmaceutical industry requires innovative experimental models. However, even if a lot of progress has been achieved by using transgenic mouse models, to date no in vivo model can reflect the complexity of human brain or reproduce a clinical context. 2D in vitro cell culture models are unable to allow the aggregation and accumulation of pathological proteins as observed in vivo. The aim of this study consists in taking advantage of the research prospects offered by induced pluripotent stem cell (iPSCs) in the field of neurosciences. iPSCs can be used to generate 3D models of differentiation also called human cerebral organoids or mini-brains (MBs). Their ability to self-organise in 3D neuroectodermic tissue leds to a complex system that mimics different human cerebral structures in which we were able to characterize the expected markers. The study of the two proteins of interest (APP and PrPC) during neural differentiation has allowed us to follow the modulation of protein expression level occurring during the in vitro development of the human MBs. In order to use this model to reproduce the protein accumulation mechanisms seen in AD, we have tested chemical inductors such as Aftin-5 in order to modulate the APP post-transcriptional pathway towards a pathological outcome. Many strategies of treatment are adopted to lead APP cleavage and Aβ generation. The production of soluble fragments Aβ38, Aβ40, Aβ42 in the supernatant of organoids has been showed using ELISA technique. The levels generated are reproducible and the increase of Aβ42/Aβ40 ratio is consistent with extrapolated data from mouse and human models thus validating our model. Analysis at the gene and protein level has been assessed in order to understand the interaction between PrPC and APP after treatment. The long-term goal consists in improving this model which is notably hampered by the absence of vascularization and the low level of maturation of the neural tissue. The main challenge in MB culture thus consists in the integration of the vascular system, and also in increasing the speed of ageing process in vitro for the study of neurodegenerative diseases. In the long term, the prospect of automating the culture of MBs would allow the use of the system for cytotoxicity testing and/or high throughput screening for the discovery of new drugs for AD

Nitrogen is necessary for the synthesis of a large number of compounds, such as amino acids, which are very important for several yeast dynamics, and for industrial purposes. This project aims at the identification and characterization of new transporters involved in the excretion of amino acids through the study of a specific gene of the MFS superfamily of transporters in eukaryotes, the DTR1 gene. In the MDR-MFS (MultiDrug Resistance-Major Facilitator Superfamily) family of proteins, up to 100 are unknown, 24 proteins are Multidrug Resistance, and may be involved in the excretion of amino acids. DTR1 is a multi-drug resistance protein with a physiological role assigned to the yeast cell where the layers of chitosan and dityrosine on the external spore wall provide greater resistance to environmental stresses. In the present study, techniques such as E. coli and S. cerevisiae transformation, PCR, RT-PCR, qPCR analysis were applied. The characterization of the DTR1 gene was carried out through its location and expression using various tools, such as the Green Fluorescent Protein by observing when it is expressed in the control of its own promoter, and a fusion with the GAL1 promoter, verifying overexpression of the protein or whether the expression is normally done. The construction of the PGAL1-DTR1-GFP cassette did not occur as expected, therefore was not possible to observe the overexpression of the DTR1 gene and its location. As for characterization on a plasmidic level, plasmids containing the GAP1 promoter were used and the promoter induced, this was possible to observe the expression analysis of the GAP1-DTR1 construction with a change of medium. Additionally, a study of the influence of glycerol and temperature, on S. cerevisiae culture was carried out where it was observed that high temperature and presence of glycerol might be stress conditions enough to DTR1 to be expressed.
O azoto é um nutriente mineral crítico em todos os organismos vivos, pois é necessário para a síntese de um grande número de compostos, incluindo hormonas, nucleotídeos e aminoácidos. Os aminoácidos são muito importantes para várias dinâmicas da levedura, como síntese de proteínas, metabolismo de hormonas, transmissão nervosa, crescimento celular, geração de energia, metabolismo do azoto e síntese de bases azotadas. Os aminoácidos também são importantes para fins industriais, como aplicações em alimentos como o aminoácidos glutamato (intensificador de sabor) ou aspartato, fenilalanina (adoçantes); para alimentação, tais como os aminoácidos lisina, metionina, treonina; e para aplicações farmacêuticas, como soluções de infusão e blocos de construção, triptofano (indutor do sono) e fenilalanina (antidepressivo). Este projeto visa a identificação e caracterização de novos transportadores envolvidos na excreção de aminoácidos por meio do estudo de um gene específico da superfamília MFS (Major Facilitator Superfamily) de compostos azotados em eucariontes, o gene DTR1. De acordo com, Sá-Correia et al., (2009) vários transportadores MDR foram identificados e estudados em diferentes organismos, particularmente aquele pertencentes à superfamília ATP-binding cassette (ABC). Um grupo de transportadores envolvidos na resistência a multidrogas menos caracterizado pertence à família MFS-MDR. Proteínas deste grupo têm vindo a receber mais atenção, maioritariamente em bactéria. Em Saccharomyces cerevisiae, a maioria dos membros desta família foi apenas descobertos aquando revelada a sequencia genómica desta levedura e caracterizados em alguns aspetos nos últimos 12 anos. A família MDR-MFS (Multidrug Resistance-Major Facilitator Superfamily) de proteínas tem aproximadamente 300 proteínas transportadoras de membrana, com até 100 sendo desconhecidas. 24 proteínas são MDR da superfamília MFS, algumas delas podem estar envolvidas na excreção de aminoácidos, como DTR1. Dentro da DHA1 (Drug: H+ antiporter family 1) que é composta por 46 proteínas, sete genes são individualmente necessários para conferir resistência à quinidina, um fármaco antiarrítmico e anti malária, entre esses genes encontram-se os genes AQR1, TPO1 e DTR1. Estes sete transportadores podem, também, proteger a célula contra outros compostos que estão normalmente ausentes no ambiente natural de células de levedura e poderão ter substratos fisiológicos específicos, onde fármacos são transportados fortuitamente ou oportunisticamente. Ainda na família DHA1, 9 proteínas mostraram ser bombas de multirresistência, 15 são provavelmente bombas de efluxo específicas para drogas e 22 são proteínas hipotéticas ou não caracterizadas. Os genes de resistência a múltiplas drogas, isto é, a aquisição simultânea de resistência a uma variedade de químicos citotóxicos, é encontrada numa grande variedade de organismos, desde bactérias a mamíferos, e isto poderá causa um severo problema clínico, principalmente no tratamento de cancros humanos e infeções de origem bacteriana e fúngica, tendo atingido proporções alarmantes nos últimos anos. DTR1 é a primeira proteína de resistência a múltiplas drogas da superfamília de facilitadores principais com um papel fisiológico atribuído na célula de levedura, mas o transporte por DTR1p pode não ser restrito ao seu substrato natural, bisformil ditirosina. As camadas de quitosana e ditirosina da parede externa dos esporos conferem maior resistência a estresses ambientais no esporo, incluindo a capacidade de passar pelo trato digestivo dos insetos, permitindo a dispersão para o meio ambiente. No presente estudo é realizada a caraterização do gene DTR1 através da sua localização e expressão com recurso a variadas ferramentas, tais como a proteína Green Fluorescent Protein, produzida pelo cnidário Aequorea victoria que emite fluorescência na zona verde do espectro visível, observando quando esta está a ser expressa no controle de seu próprio promoter. Uma fusão com o promotor Gal1, um operão procariótico que codifica as enzimas necessárias para o metabolismo da galactose, para verificar a sobrexpressão da proteína ou se a expressão é feita normalmente como no seu estado natural. Com essas fusões podemos analisar se a proteína está bem expressa, superexpressa e onde ela ocorre na célula e, usando plasmídeos identificados como GAP1 e UGA4 ambos contendo o promotor GAP1, ativado pela falta de azoto, para observação do comportamento do gene em estudo. Essas ferramentas contribuem para a caracterização a nível genómico e plasmídico da proteína de interesse, DTR1, para compreender sua função e localização no genoma da levedura. O objetivo principal deste trabalho foi construir uma cassete combinando primers para o plasmídeo, primeiro e para localização do DTR1 foi utilizado o plasmídeo PKT140, que contém a proteína GFP e resistência à canamicina. Os primers construídos são homólogos à proteína GFP, no caso do F5, e à resistência à canamicina, no caso do primer R3. A estratégia adotada foi a utilização de um fragmento do plasmídeo contendo os primers, GFP e a resistência à canamicina, que foi amplificado com um tamanho esperado de 2515 pares de bases e transformado em S. cerevisiae. A tag GFP foi aplicada a jusante do gene de interesse, DTR1. A canamicina foi usada como marca de seleção no momento da transformação e a integração no genoma da levedura foi concluída por recombinação homóloga. Para compreender se a proteína de interesse, DTR1, está a ser superexpressa, um fragmento do plasmídeo PGAL1 foi usado, novamente os primers foram construídos contendo parte de DTR1 e o promoter Gal1 no caso do iniciador R2 e parte de DTR1 e resistência à nourseotricina em o caso do primer F4. Para análise deste estudo recorreu-se a técnicas como transformação em E. coli e em S. cerevisiae, digestão com recurso a enzimas de restrição, PCR, RT-PCR (Reverse Transcriptase PCR), análise de expressão por qPCR (Quantitative Real Time PCR), Cross-feeding e ainda Western Blotting. O fragmento do plasmídeo mais primers foi amplificado por PCR com 1850 pares de bases e a integração no genoma da levedura é feita por recombinação homóloga. A nível funcional concluímos que não foi possível a construção da cassete PGAL1-DTR1-GFP tendo sido efetuadas várias tentativas da mesma não tendo sido possível a observação da sobrexpressão do gene DTR1 e a sua localização. A nível plasmídico foi possível uma observação de analise de expressão de diferentes estudos de comportamento da construção GAP1-DTR1 com alteração de meio. Foi ainda feito um estudo de influência do glicerol e temperatura em cultura de S. cerevisiae em meio mínimo. Tendo em conta que o gene DTR1 faz parte da reprodução de S. cerevisiae através esporulação em condições de stress, verificou-se que uma temperatura de 30ºC e glicerol 10% poderão representar condições de stress suficientes para que este gene seja expresso.

Ectopic and over-expression of G protein-coupled receptor (GPCR) have been reported to induce tumor formation. Mas protein is a member of GPCR family and was originally isolated from human epidermoid carcinoma. It was demonstrated that mas mRNA was abundantly expressed in human and rat brains by in situ hybridization and RNase protection assays. However, cellular mechanism that leads to such tumorigenic transformation is still an open question.
In order to identify the cellular mechanism of mas-induced tumor formation, a full-length mas cDNA was cloned into a mammalian expression vector pFRSV with dihydrofolate reductase gene as a selection marker. Detailed analyses of mas-transfected cell lines by Southern blot, Northern blot and tumorigenicity assay indicated that tumorigenicity of mas-transfected cells depended on the sites of chromosomal integration and the levels of mas expression. These results suggest that overexpression of mas is not sufficient to induce tumor formation. In line with the ability of mas-transfected cells Mc0M80 to form solid tumor in nude mice, MTT cell proliferation assay indicated that the mas-transfected cells Mc0M80 proliferated faster than vector-transfected cells. Moreover, mas-transfected cells Mc0M80 exhibited significantly increased anchorage-independent growth. Furthermore, mas-transfected cells Mc0M80 showed higher percentage cells in G2/M phase but lower in S-phase in comparison with vector-transfected cells.
Interestingly, Southern blot analysis of individual xenografted tumor tissue indicated that tumor was composed of cells not only derived from injected mas-transfected CHO cells but also cells from mouse tissues. The presence of mouse stromal cells in the tumor was confirmed by immunohistochemistry and in situ hybridization. Previously our laboratory had identified some up- and down-regulated genes in mas-transfected cells by fluorescent differential display (FluoroDD). Northern blot showed that these differential expressed genes were up- or down-regulated in mas-transfected cells and tumor samples, which might play an important role in cancerous growth.
Taken together, these results suggest that over-expression of GPCR mas up-regulated tumor-related genes, resulting in promoting excessive cell growth and tumorigenic transformation. In addition, when the tumor mass formed they secreted some growth factor(s) which altered the migration of mouse stromal cells into tumor mass. The interactions of transformed cells and stromal cells further aggravate the tumorigenicity process.
To complement our fluorescent differential display study and to compare changes of gene expression when transformed cells were exposed to the microenvironment in nude mice, protein expression profiles of mas over-expressing cells as well as tumor tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The 2D-PAGE analysis showed that a similar but distinct protein expression profiles in mas-transfected cells and in mas-induced tumor. Mass spectrometry analysis identified several cancerous growth-related proteins and they are involved in processes such as cell signaling, energy metabolism, transcription and translation and cytoskeleton organization.
Lin Wenzhen.
"December 2005."
Adviser: Cheung Wing Tai.
Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6381.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (p. 222-240).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

In a phage binding assay, phage clone (3p5A190) expressing a surrogate mas ligand displayed punctate binding and were internalized in cell expressing native mas and GFP-tagged variants. However, the number of bound and internalized phages in cells expressing mas-GFP was substantially less than the cells expressing mas-(Gly10Ser5)GFP and native mas. In parallel, biotinylation experiment quantitatively showed that the extent of mas-(Gly10Ser 5)-GFP translocation was higher than that of mas-GFP. Consistently, cells expressing mas-(Gly10Ser5)-GFP and native mas showed a rapid and sustained increase of intracellular calcium levels upon MBP7 stimulation. By contrast, cells expressing mas-GFP only response to higher concentration of MBP7 challenge and showed a delayed increase of intracellular calcium level. Moreover, cells expressing native mas had a higher proportion (80%) of cells responsive to MBP7 stimulation; in contrast to only 10∼20% of cells expressing mas fusion proteins.
MBP7-like motif was identified in human facilitative GLUT1 and GLUT7 indicating that mas might interact with glucose transporter (GLUT) and regulate cellular glucose uptake. GLUT4 was found to be expressed endogenously in the CHO cell by RT-PCR, but expression of insulin receptor was not detectable. Although no statistical difference was detected in basal glucose uptake among control cells Vc0M80 and cells with different levels of mas expression, cells expressing mas-(Gly10Ser5)-GFP showed a high glucose uptake in response to insulin. Furthermore, basal 2-DOG uptake in Mc0M80 cells was not affected by pretreatment with various kinase inhibitors or transient expression of Rho variants. By contrast, MBP7 was found to induce a significant elevation of glucose uptake specifically in Mc0M80 cells transiently transfected with GLUT1.
Orphan G protein-coupled receptor (GPCR) mas was initially isolated from a human epidermal carcinoma. Previous study from our lab identified a surrogate ligand---MBP7 (mas binding peptide 7) for mas, and suggested that GFP tagging might affect the receptor activity of mas. In this project, three stable CHO cell lines expressing native mas, mas-GFP and mas-(Gly10Ser 5)-GFP were used to characterize receptor activity of mas.
To summarize, direct GFP tagging at the C-terminus of mas decreased its interactions with ligand and downstream signaling molecules. Partial recovery of mas receptor activity by adding a peptide linker was confirmed by phage binding, membrane fusion protein translocation and calcium response. In addition, mas was possibily coupled with GLUT1 to affect cellular glucose uptake via signaling pathways yet to be fully characterized.
Sun, Jingxin.
Adviser: Cheung Wing Tai.
Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0104.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 150-170).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.

Tsang Sup-Yin.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 176-185).
Abstracts in English and Chinese.
Acknowledgments --- p.1
Abstract --- p.2
摘要 --- p.4
List of Abbreviation --- p.6
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- Isolation and activation of mas oncogene --- p.11
Chapter 1.2 --- Amino acid sequence of mas oncogene --- p.14
Chapter 1.3 --- Expression of mas oncogene --- p.18
Chapter 1.4 --- Possible physiological role of mas oncogene --- p.20
Chapter 1.5 --- Gene related to mas family --- p.23
Chapter 1.6 --- Aims of study --- p.26
Chapter Chapter 2 --- Over-expression of mas oncogene
Chapter 2.1 --- Introduction --- p.28
Chapter 2.2 --- Materials and Methods --- p.29
Chapter 2.2.1 --- Materials --- p.30
Chapter 2.2.1.1 --- Chemicals --- p.30
Chapter 2.2.1.2 --- Enzyme --- p.30
Chapter 2.2.1.3 --- DNA Purification Kit --- p.31
Chapter 2.2.1.4 --- Others --- p.31
Chapter 2.2.2 --- Methods --- p.31
Chapter 2.2.2.1 --- Strategy of construct preparation --- p.31
Chapter 2.2.2.2 --- "Preparation of linearized vector, pFRSV" --- p.32
Chapter 2.2.2.2.1 --- Cloning of vectors --- p.32
Chapter 2.2.2.2.2 --- Restriction enzyme digestion and DNA dephosphorylation --- p.34
Chapter 2.2.2.2.3 --- DNA purification by agarose gel electro-elution --- p.34
Chapter 2.2.2.3 --- Preparation of pFRSV/mas construct --- p.35
Chapter 2.2.2.3.1 --- PCR amplification --- p.35
Chapter 2.2.2.3.2 --- Restriction enzyme digestion --- p.36
Chapter 2.2.2.4 --- Ligation and analysis --- p.37
Chapter 2.2.2.5 --- Purification of DNA by cesium chloride --- p.38
Chapter 2.2.2.5.1 --- Large-scale bacterial culturing --- p.38
Chapter 2.2.2.5.2 --- Ethanol precipitation --- p.39
Chapter 2.2.2.5.3 --- Cesium chloride purification --- p.39
Chapter 2.2.2.5.4 --- Removal of DNA dye by dialysis and ethanol precipitation --- p.40
Chapter 2.2.2.6 --- Transfection by electroporation --- p.41
Chapter 2.2.2.7 --- Screening for the stably transfected cells --- p.42
Chapter 2.2.2.8 --- RT-PCR analysis of the mas transfectant --- p.43
Chapter 2.2.2.8.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.43
Chapter 2.2.2.8.2 --- Reverse transcription of the total RNA into cDNA --- p.44
Chapter 2.2.2.8.3 --- Analysis of the transfected mas expression by PCR --- p.44
Chapter 2.2.2.8.4 --- Analysis of the transfected DHFR expression by PCR --- p.45
Chapter 2.2.2.8.5 --- Analysis of endogenous GAPDH expression by PCR --- p.46
Chapter 2.2.2.9 --- Amplification of mas transgene by using methotrexate --- p.47
Chapter 2.2.2.9.1 --- Amplification by low dosage MTX treatment --- p.47
Chapter 2.2.2.9.2 --- Amplification by high dosage MTX treatment --- p.49
Chapter 2.2.2.10 --- Southern blot analysis --- p.50
Chapter 2.2.2.10.1 --- Preparation of DIG-labelled mas probe --- p.51
Chapter 2.2.2.10.2 --- Preparation of DIG-labelled DHFR probe --- p.51
Chapter 2.2.2.10.3 --- Preparation of DIG-labelled GAPDH probe --- p.52
Chapter 2.2.2.10.4 --- Isolation of Genomic DNA from the mas transfectants by DNAzol® Reagent
Chapter 2.2.2.10.5 --- Enzymatic restriction of genomic DNA and Gel electrophoresis --- p.54
Chapter 2.2.2.10.6 --- DNA transferring to positive charged Nylon membrane --- p.54
Chapter 2.2.2.10.7 --- Pre-hybridization and hybridization --- p.56
Chapter 2.2.2.10.8 --- Post-hybridization washing and blocking --- p.56
Chapter 2.2.2.10.9 --- Detection --- p.57
Chapter 2.2.2.11 --- Northern blot analysis --- p.57
Chapter 2.2.2.11.1 --- Preparation of the agarose gel containing formaldehyde --- p.58
Chapter 2.2.2.11.2 --- Preparation of the RNA sample --- p.58
Chapter 2.2.2.11.3 --- Gel electrophoresis and transferring --- p.59
Chapter 2.2.2.11.5 --- Pre-hybridization and hybridization --- p.60
Chapter 2.2.2.11.4 --- Post-hybridization washing and blocking --- p.60
Chapter 2.2.2.11.6 --- Detection --- p.61
Chapter 2.2.2.11.7 --- Stripping and rehybridization --- p.61
Chapter 2.3 --- Results --- p.62
Chapter 2.3.1 --- RT-PCR analysis of gene expression in the stably transfectant --- p.62
Chapter 2.3.2 --- Morphology of the mas trasnfectant --- p.64
Chapter 2.3.3 --- Determination of mas gene copy number by Southern blot analysis in the mas transfectants --- p.66
Chapter 2.3.4 --- Northern blot analysis of the transcriptional level of mas transcriptsin mas transfectants --- p.76
Chapter 2.4 --- Discussion --- p.87
Chapter Chapter 3 --- In vivo study of physiological effect of over-expression of mas
Chapter 3.1 --- Introduction --- p.92
Chapter 3.2 --- Materials and Methods --- p.93
Chapter 3.2.1 --- Materials --- p.93
Chapter 3.2.2 --- Methods --- p.93
Chapter 3.2.2.1 --- Cell culture --- p.93
Chapter 3.2.2.2 --- Subcutaneous injection of nude mice --- p.94
Chapter 3.2.2.3 --- Isolation of the total RNA from the tumor tissues --- p.95
Chapter 3.2.2.4 --- Northern blot analysis --- p.96
Chapter 3.3 --- Results --- p.96
Chapter 3.3.1 --- Tumorgenicity assay of mas oncogene in nude mice --- p.96
Chapter 3.3.2 --- Northern blot analysis of mas expression in the tumor tissues --- p.103
Chapter 3.4 --- Discussion --- p.109
Chapter Chapter 4 --- Fluorescent differential display analysis of mas transfectants
Chapter 4.1 --- Introduction --- p.111
Chapter 4.2 --- Materials and Methods --- p.112
Chapter 4.2.1 --- Materials --- p.112
Chapter 4.2.1.1 --- Chemicals --- p.112
Chapter 4.2.1.2 --- Enzyme --- p.113
Chapter 4.2.1.3 --- Kits --- p.113
Chapter 4.2.1.4 --- Others --- p.114
Chapter 4.2.2 --- Methods --- p.114
Chapter 4.2.2.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.114
Chapter 4.2.2.2 --- DNase I treatment --- p.115
Chapter 4.2.2.3 --- Reverse transcription (RT) and non-fluorescent PCR --- p.116
Chapter 4.2.2.4 --- Reverse transcription and fluorescent differential display-PCR --- p.118
Chapter 4.2.2.5 --- High resolution fluorescent differential display (Fluoro DD) gel --- p.118
Chapter 4.2.2.6 --- Gel band excision of differentially expressed cDNA fragments --- p.120
Chapter 4.2.2.7 --- Gel band reamplification --- p.120
Chapter 4.2.2.8 --- Subcloning of reamplified cDNA fragments --- p.121
Chapter 4.2.2.9 --- Purification of plasmid DNA from recombinant clones for reverse dot blot analysis --- p.122
Chapter 4.2.2.10 --- Reverse dot blot analysis --- p.123
Chapter 4.2.2.10.1 --- Preparation of cDNA dot blot --- p.123
Chapter 4.2.2.10.2 --- Preparation of DIG-labeled cDNA library probes --- p.124
Chapter 4.2.2.10.3 --- Hybridization --- p.126
Chapter 4.2.2.11 --- Northern blot analysis --- p.127
Chapter 4.3 --- Results --- p.128
Chapter 4.3.1 --- Fluorescent differential display (FluoroDD) --- p.128
Chapter 4.3.2 --- Reverse dot blot analysis --- p.135
Chapter 4.3.3 --- DNA sequencing analysis of the clones --- p.141
Chapter 4.3.4 --- Confirmation of differential display pattern of the subclones by Northern blot analysis --- p.160
Chapter 4.4 --- Discussion --- p.166
Chapter Chapter 5 --- General Discussion
Chapter 5.1 --- General model for mos-induced tumor formation --- p.169
Chapter 5.2 --- Future aspect --- p.174
References --- p.176
Appendix I Buffer composition --- p.186
Appendix II Sequences of fluoroDD TMR-Anchored primers and arbitrary primers --- p.190

Bikkavilli Rama Kamesh.
"August 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 212-223)
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.

碩士
高雄醫學大學
醫學研究所
96
The transcription factors C/EBP (CCAAT/Enhancer Binding Protein) family is a highly conserved family of leucine zipper composed of six members: Cebpa, Cebpb, Cebpg, Cebpd, Cebpe and Cebpz. Cebpd originally was identified as an inflammatory response gene. Cebpd is expressed in a variety of tissues and cell types and involved in the control of cellular proliferation, differentiation, metabolism, inflammation and maintenance of genomic stability. In our previous studies, we had demonstrated that Cebpd knockout (KO) mouse embryonic fibroblasts (MEFs) exhibited significant higher resistance to cisplatin through altering the p53 and Erk signaling pathways. This suggests that Cebpd could serve as a mediator in response to genotoxic agents. Methyl methanesulophonate (MMS) is an alkylating agent. The removal of MMS-induced DNA adducts is mainly through base excision repair. In this study, we found that Cebpd KO MEFs (NKO) showed higher resistance to MMS when compared to wild type. In addition, Cebpd protein and p53 protein activation (phospho-Ser-15) can be induced by MMS within five hours in NWT. p21 protein, a well-known p53 target gene, was also induced at the same kinetics. However, Cebpd and p53 mRNA level were not induced after MMS treatment. The phosphorylation of p38 can also be induced by MMS, but there was no difference whether Cebpd existence or not. Phosphorylated p38 can directly activate p53. These data indicate that Cebpd may play an important role in regulating of apoptosis and DNA repair in MEFs. The molecular mechanism by which Cebpd might involve in MMS-induced DNA damage response needs to be explored. This study reveals a potential role of Cebpd in MMS-induced DNA damage response in MEFs.

Tang Wai-man.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 142-147).
Abstract also in Chinese.
Abstract --- p.i
摘要 --- p.iii
Acknowledgement --- p.v
Lists of Abbreviations --- p.vi
Table of Contents --- p.vii
Chapter Chapter 1: --- Introduction
Chapter 1.1 --- Isolation of Mas Oncogene --- p.1
Chapter 1.2 --- Distribution of Mas Oncogene..........…… --- p.3
Chapter 1.3 --- Developmental Expression of Mas Oncogene --- p.5
Chapter 1.4 --- Study of Mas-deficient Mice --- p.7
Chapter 1.5 --- Signal Transduction of Mas Oncogene --- p.8
Chapter 1.6 --- Other Family Member of Mas Oncogene --- p.9
Chapter 1.7 --- Mas and Angiotensin Receptor --- p.11
Chapter 1.8 --- Angiotensin Receptors
Chapter 1.8.1 --- Classification of Angiotensin AT1 Receptor --- p.14
Chapter 1.8.2 --- Cloning of Angiotensin Receptor --- p.15
Chapter 1.9 --- Expression of Angiotensin Receptor
Chapter 1.9.1 --- Physiological Factors --- p.17
Chapter 1.9.2 --- Cis-regulatory Elements
Chapter 1.9.2.1 --- Organization and Regulatory Elements of AT1 Receptor --- p.19
Chapter 1.9.2.2 --- Expression of AT1a Receptor Promoter was Induced by AP-1 and GATA-4 in Pressure Overload Model --- p.20
Chapter 1.9.2.3 --- AT1a Receptor Reveals Three Glucocorticoid Responsive Elements --- p.22
Chapter 1.10 --- Signal Transduction of Angiotensin Receptor --- p.22
Chapter 1.11 --- Aim of Project --- p.25
Chapter Chapter 2: --- Mas Oncogene in AR4-2J cells
Chapter 2.1 --- Introduction --- p.26
Chapter 2.2 --- Materials and Methods
Chapter 2.2.1 --- Materials
Chapter 2.2.1.1 --- Reagents --- p.27
Chapter 2.2.1.2 --- Enzymes --- p.27
Chapter 2.2.1.3 --- DNA Purification Kits --- p.28
Chapter 2.2.1.4 --- Materials and Antibodies for Western Blot --- p.28
Chapter 2.2.1.5 --- Others --- p.28
Chapter 2.2.2 --- Restriction Enzyme Digestion --- p.29
Chapter 2.2.3 --- Agarose Gel Electrophoresis --- p.29
Chapter 2.2.4 --- DNA Extraction and Purification --- p.29
Chapter 2.2.5 --- Plasmid Vector Modification and DNA Ligation --- p.30
Chapter 2.2.6 --- Bacterial Transformation --- p.31
Chapter 2.2.7 --- Preparation of Plasmid DNA
Chapter 2.2.7.1 --- Minipreps --- p.32
Chapter 2.2.7.2 --- Midipreps and Maxipreps --- p.33
Chapter 2.2.8 --- Genomic DNA Extraction From Tissue and Cell Culture --- p.34
Chapter 2.2.9 --- RT-PCR Cloning of Mas Oncogene --- p.35
Chapter 2.2.10 --- Construction of Full Length Mas cDNA into pBluescript® II SK Vector --- p.38
Chapter 2.2.11 --- Southern Blot Analysis
Chapter 2.2.11.1 --- Preparation of DIG-labeled Mas Probe --- p.38
Chapter 2.2.11.2 --- Enzyme Restriction of Genomic DNA --- p.39
Chapter 2.2.11.3 --- Transferring DNA to Nylon Membrane --- p.40
Chapter 2.2.11.4 --- Prehybridization and Hybridization --- p.40
Chapter 2.2.11.5 --- Post-hybridization Washes and Blocking --- p.41
Chapter 2.2.11.6 --- Detection --- p.41
Chapter 2.2.12 --- DNA Sequencing
Chapter 2.2.12.1 --- Manual Sequencing --- p.42
Chapter 2.2.12.2 --- Autosequencing --- p.43
Chapter 2.2.12.3 --- Sequencing Primers --- p.44
Chapter 2.2.13 --- Cell Culture --- p.45
Chapter 2.2.14 --- Protein Assay by Modified Lowery --- p.46
Chapter 2.2.15 --- SDS-PAGE and Western Blot Analysis --- p.47
Chapter 2.3 --- Results --- p.49
Chapter 2.4 --- Discussion --- p.60
Chapter Chapter 3: --- Analysis of Transfected Mas Cell Lines
Chapter 3.1 --- Introduction --- p.61
Chapter 3.2 --- Materials and Methods
Chapter 3.2.1 --- Materials --- p.62
Chapter 3.2.2 --- Cell Culture and Transfection
Chapter 3.2.2.1 --- Cell Culture --- p.62
Chapter 3.2.2.2 --- Transfection Optimization --- p.62
Chapter 3.2.2.3 --- Fluorescent SEAP Assay --- p.63
Chapter 3.2.2.4 --- Transient Transfection --- p.64
Chapter 3.2.2.5 --- Stable Cell Line Construction --- p.64
Chapter 3.2.3 --- Protein Assay ESL --- p.65
Chapter 3.2.4 --- SDS-PAGE and Western Blot Analysis --- p.65
Chapter 3.2.5 --- Preparation of an AT1a Receptor Internal Standard for Quantitative RT-PCR Analysis
Chapter 3.2.5.1 --- Preparation of an AT1a Receptor cDNA by RT-PC --- p.66
Chapter 3.2.5.2 --- Cloning of AT1A Receptor cDNA into pBluescript® II SK Vector --- p.67
Chapter 3.2.5.3 --- Autosequence of pBluescript® II SK Vector/AT1AR --- p.68
Chapter 3.2.5.4 --- Preparation of 100 bp Deleted AT1a Receptor cDNA by RT- PCR --- p.68
Chapter 3.2.5.5 --- Cloning of Deleted AT1a R cDNA into pCAPs Vector --- p.71
Chapter 3.2.6 --- Construction of Full Length Mas cDNA into pOPRSVI/MCS Operator Vector --- p.71
Chapter 3.2.7 --- Preparation of an Mas Internal Standard for Quantitative RT-PCR Analysis
Chapter 3.2.7.1 --- Preparation of 100 bp Deleted Mas cDNA by RT- PCR --- p.72
Chapter 3.2.7.2 --- Cloning of 100 bp Deleted Mas cDNA into pCAPs Vector (Mas/pCAPs) --- p.73
Chapter 3.2.8 --- Quantitative RT-PCR Analysis of AT1A R Expression --- p.74
Chapter 3.2.9 --- Quantitative RT-PCR Analysis for the Expression of Mas --- p.74
Chapter 3.3 --- Results --- p.76
Chapter 3.4 --- Discussions --- p.100
Chapter Chapter 4: --- Cloning of AT1A Receptor Promoter
Chapter 4.1 --- Introduction --- p.104
Chapter 4.2 --- Materials and Methods
Chapter 4.2.1 --- Materials --- p.105
Chapter 4.2.2 --- Genomic DNA Extraction From Rat Pancreas --- p.105
Chapter 4.2.3 --- "Nest PCR Amplification of 3.2, 2.8 and 1.4kb AT1a Receptor Promoter" --- p.105
Chapter 4.2.4 --- PCR Amplification of 2.2 kb Aproximal Portion of AT1a Receptor Promoter --- p.107
Chapter 4.2.5 --- Construction of PCR Fragment of Angiotensin Receptor Promoter into Various Vector --- p.108
Chapter 4.2.5.1 --- pSEAP2-Basic --- p.108
Chapter 4.2.5.2 --- pBluescript® II SK Vector --- p.109
Chapter 4.2.5.3 --- PCR Cloning Kit (pCAPs vector) --- p.109
Chapter 4.2.5.4 --- PCR-TRAP Cloning System --- p.109
Chapter 4.2.6 --- Direct PCR Analysis --- p.110
Chapter 4.2.7 --- Autosequencing of PCR Fragment of AT1A Receptor Promoter --- p.111
Chapter 4.3 --- Results --- p.114
Chapter 4.4 --- Discussions --- p.130
Chapter Chapter 5: --- General Discussion --- p.131
Chapter Appendix 1 --- Composition of Solutions --- p.133
Chapter Appendix 2 --- Published Abstract --- p.141
References --- p.142

Understanding the structure-function relationship of proteins such as G protein-coupled receptors (GPCRs) requires detailed information on their three-dimensional (3D) structure and dynamics. Recent progress in the structural elucidation of receptor-G protein or -arrestin complexes yields valuable starting points to studying their function and dynamics. I investigated the dynamic process of β2 adrenergic receptor-G protein recognition and binding using molecular dynamics (MD) simulations. MD simulation is a well-established technique to monitor the time-resolved motions of proteins and is used to investigate the dynamics of GPCRs. In order to start MD simulations, the structures that are often incompletely resolved have to be further completed. Finally, the results from computational analysis have to be subjected to experimental validation. To promote broad applicability of that workflow, I automated critical steps such as modeling of missing segments or interactive analysis and visualization of the results of MD simulations. Web-tools allow researchers besides different methodological expertise to apply unfamiliar techniques on their biological systems of interest without the need to download software or databases. To facilitate usage, I included visualization allowing intuitive understanding and analysis. We developed the NGL molecular web-viewer, which accesses hardware-accelerated graphics through WebGL. This and other recent developments, reviewed by Mwalongo and others, opened up new possibilities for web molecular graphics and render- ing techniques, superseding plugin- and Java-based viewers and making them comparable with desktop tools, such as PyMOL, VMD, or UCSF Chimera. Here, I implemented and applied tools for interactive modeling of missing segments into single-particle cryo-electron microscopy (cryo-EM) density maps, for interactive analysis of structure-functional dynamics, and more general to promote interdisciplinary research. To allow non-expert users access to bioinformatics methods and biophysical techniques, all tools were generated as web-services allowing interactive analysis and visualization.:Introduction 1 GPCRs as a prime example in structural and pharmaceutical biology 2 Modeling missing segments into cryo-EM density maps 3 Research goal I: Web service for modeling of missing segments into cryo-EM density maps 4 From static to dynamics to investigate GPCR function 5 The complexity of sharing MD simulations 6 Research goal II: Interactive visual sharing of MD simulations Manuscripts 1 FragFit: a web-application for interactive modeling of protein segments into cryo-EM density maps 2 MDsrv: viewing and sharing molecular dynamics simulations on the web. 3 Bringing Molecular Dynamics Simulation Data into View Summary 1 Modeling of missing fragments into cryo-EM density maps 2 InteractiveanalysisofMDsimulationsontheweb . . . . . . References Appendix A Additional Manuscripts 1 SL2: an interactive webtool for modeling of missing segments in proteins 2 GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors. 3 A fragment based method for modeling of protein segments into cryo-EM density maps 4 Structural Insights into the Process of GPCR-G Protein Complex Formation 5 Sharing Data from Molecular Simulations Appendix B Darstellung des eigenen Beitrags Selbständigkeitserklärung Curriculumvitae & Publikationen Danksagung

Damage to actively transcribed genes is repaired at a faster rate than damage to non-transcribed genes due to the activity of proteins called transcription repair coupling factors. These proteins recognize RNA polymerases that have stalled on damaged DNA, remove them, and recruit DNA repair proteins to the site of the damage. In bacteria one protein performs all of those functions, Mfd. We are investigating the regulation of Mfd's activity and we hypothesize that the opening and closing of the interface between the protein's two principal domains, MfdN (residues 1-450) and MfdC (residues 473-1148), toggles Mfd between active and inactive states. We have solved the crystal structure of MfdN by molecular replacement. Its structure superimposes well with that same region from the crystal structure of the full length Mfd protein (Deaconescu et al 2006), with an rmsd of 1.1 Å. The fold of MfdN appears to be independent of MfdC and the linker region (451-472), which is too disordered to be seen in the crystal structure. We have probed the stability of the N and C terminal regions of Mfd expressed as separate constructs, and the effect of disrupting the interface between them on the properties of Mfd. We believe that MfdN exerts a stabilizing influence on structure of full length Mfd, and that it has an inhibitory effect on the protein's activity through its binding interactions with MfdC.

博士
高雄醫學大學
醫學研究所博士班
103
TGF-β1 and high glucose are pivotal factors in diabetic nephropathy (DN) and they induce irreversible renal fibrosis. ACE2-Ang-(1-7)-Mas pathway is also a key mediator in DN. In previous studies, ACE2 and Mas were decreased in DN, meanwhile, high glucose inhibited ACE2 and Mas mRNA in kidney cells. However, the influence of TGF-β1 on ACE2-Ang-(1-7)-Mas pathway remains unclear. Moverover, we also investigated that the interaction between TGF-β1, high glucose and ACE2-Ang-(1–7)-Mas in NRK-52E cells. In our research, the results showed that high glucose inhibited ACE2, Mas and Ang-(1-7) production through TGF-β pathway. TGF-β1 also reduced ACE2-Ang-(1-7)-Mas pathway. Further, Ang-(1-7) inhibited TGF-β1 mRNA expression through Mas receptor、JAK2-STAT3 and PI3K-AKT pathways. However, Ang-(1–7) alone did not attenuate TGF-β1 and high glucose-induced fibronectin and collagen IV protein expression. In contrast, Ang-(1–7) in combination with Mas overexpression attenuated TGF-β1-induced fibronectin. We suggested that there is a negative feedback function between TGF-β1 and ACE2-Ang-(1-7)-Mas pathway. In animal experiment, we detected serum protein of rats with DN by using proteomic techniques. The results showed that the level of serum DBP was changed in diabetic rats. Therefore, our research focus on the role of DBP in DN. First, we confirmed that serum DBP was phosphorylated in diabetic rats. Meanwhile, DBP levels were increased in kidney and liver of diabetic rats, and the pI of DBP was shifted to the lower values. Next, we confirmed that high glucose increased DBP expression in NRK-49F renal fibroblasts and Clone-9 hepatocytes. Finally, we found that vitamin D attenuated of HG-induced renin, collagen IV and fibronectin protein expression in NRK-49F cells, however, DBP silencing only attenuated vitamin D-induced attenuation of HG-induced renin protein expression. In conclusion, both of TGF-β1 and high glucose inhibited the renoprotective effects of ACE2-Ang-(1-7)-Mas pathway, resulting in high level of Ang II. High level of Ang II induced renal fibrosis. Renin is a important enzyme for RAS activation, and DBP is required for vitamin D-induced attenuation of HG-induced renin. Therefore, the efficient inhibitory effect of vitamin D on RAS activation is very important.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
The large majority of receptors in human organism belongs to the superfamily of G protein-coupled receptors (GPCR). This superfamily is constituted of eleven families with distinct ligands and physiological roles, meaning they are a good drug target. Many GPCRs do not have any known ligand being denominated as orphan receptors. Over the past years several compounds were found as ligands for these receptors using reverse pharmacology. One of the families belonging to the GPCR superfamily is the Mas-related gene X (MrgX) receptor family. The MrgX2 receptor is a member of this family and is implicated in nociception and in inflammatory process. Cortistatin-14 has been reported as a natural ligand, but other agonists have also been identified. Despite this, no MrgX2 antagonist has yet been described. In this study two compound libraries were screened using β-arrestin assay in order to identify antagonists for the MrgX2 receptor. From the 1560 screened compounds one demonstrated a possible antagonistic activity: menadione. This is an analogue of vitamin K. Further studies must be performed in order to assess the mechanism of the antagonistic effect and the specificity of menadione. This molecule may serve as a lead structure for the development of more potent MrgX2 antagonists.
A grande maioria dos recetores no organismo humano pertence à superfamília dos recetores acoplados à proteína G (GPCR). Esta superfamília é constituída por onze famílias com ligandos e funções fisiológicas distintas, conferindo-lhes especial interesse como alvos terapêuticos. Muitos GPCRs não têm ligando conhecido e são denominados recetores órfãos. Nos últimos anos muitos compostos foram classificados como ligandos destes recetores, usando farmacologia reversa. Uma das famílias que pertence à superfamília GPCR é a dos recetores Masrelated gene X (MrgX). O recetor MrgX2 é um membro desta família que participa na nocicepção e no processo inflamatório. A cortistatina-14 foi identificada como o seu ligando natural, mas outros agonistas também têm sido reportados. No entanto nenhum antagonista foi descrito até à data. Neste estudo duas bibliotecas de compostos foram testadas usando o ensaio da β-arrestina com o objetivo de encontrar um antagonista para o recetor MrgX2. Dos 1560 compostos testados um mostrou possível atividade antagonista: a menadiona. Esta é um análogo da vitamina K. É ainda necessário o desenvolvimento de estudos posteriores que permitam verificar o mecanismo do efeito antagonista e a especificidade da menadiona. Esta molécula poderá servir como estrutura modelo para o desenvolvimento de antagonistas mais potentes para o recetor MrgX2.
Rheinische Friedrich-Wilhelms Universität Bonn, Pharmazeutisches Institut

Peroxisomes are single membrane bound organelles involved in crucial cellular metabolic functions. They were noticed for the first time in the 1960s and, along the years, their importance for the cellular homeostasis has been increasingly highlighted. Peroxisomes and mitochondria cooperate in several cellular metabolic processes and more recently were found to have a complementary role in the antiviral innate immune response. These two organelles also share many proteins including the fission machinery key proteins MFF, FIS1 and DLP1 and the antiviral signaling protein MAVS. As the proper function of these organelles strongly depends on the capacity to adequate their shape and cellular localization in accordance with the cellular needs, and thus on the correct regulation of membrane fission events, in this work we evaluated the role of the peroxisomal and mitochondrial fission machinery, specially the key DLP1 adaptors MFF and FIS1 in the antiviral immune response against one of the most spread viruses in the community: the human cytomegalovirus (HCMV). In line with this, we started this work by characterizing the peroxisomal fission machinery and by distinguishing different roles of key components shared with mitochondria (MFF e FIS1). The role of these proteins in the peroxisomal antiviral signaling against HCMV was afterwards analyzed in detail. Our results strongly indicate that MFF plays a crucial role at the regulation of peroxisomal fission whereas FIS1 significantly impacts mitochondrial fission events. In addition, we found that MFF interacts with the HCMV viral protein vMIA and that it is essential to vMIA´s function. MFF seems to, thus, play a crucial role in HCMV´s infection. Altogether, these results empathize the importance of peroxisomal fission machinery for the RLR-mediated antiviral defense and may lead to the discovery of novel peroxisome-dependent mechanisms, which can ultimately be used as targets for antiviral therapy.
Os peroxissomas são organelos celulares de membrana simples que desempenham funções metabólicas cruciais. Eles foram descritos pela primeira vez nos anos 60 e, ao longo dos anos, sua importância para a homeostasia celular tem sido cada vez mais destacada. Ao nível celular, os peroxissomas e as mitocôndrias cooperam em vários mecanismos metabólicos e recentemente foi descoberto que também têm funções complementares ao nível da resposta imune antiviral. Estes organelos também partilham várias proteínas, incluindo as proteínas chave das suas maquinarias de divisão MFF, FIS1 e DLP1 bem como a proteína antiviral MAVS. Como a correta função destes organelos depende em grande parte da capacidade de adequar a sua morfologia e localização celular de acordo com as necessidades das células e, por isso, da correta regulação dos eventos de fissão membranar, neste trabalho decidimos avaliar o papel da maquinaria de divisão peroxissomal e mitocondrial, especificamente dos adaptadores chave da DLP1, MFF e FIS1 na resposta antiviral contra um dos vírus mais disseminados na comunidade: o citomegalovírus humano (HCMV). Assim, iniciámos este trabalho com a caracterização da maquinaria de divisão peroxissomal a com a distinção de diferentes funções das proteínas de fissão partilhadas com as mitocôndrias (MFF e FIS1). Seguidamente, analisámos em detalhe a função destas proteínas na sinalização antiviral peroxissomal na defesa contra o HCMV. Os nossos resultados indicam que a MFF desempenha um papel crucial na regulação da fissão peroxissomal, enquanto que a FIS1 parece ter maior impacto na divisão mitocondrial. Além disso, foi descoberto que a MFF interage com a proteína viral vMIA, e assim parece desempenhar um papel crucial na infeção pelo HCMV. No seu conjunto, estes resultados enfatizam a importância da maquinaria de divisão peroxissomal na defesa antiviral mediada pelos RLR e poderá levar, em última instância, à descoberta de novos mecanismos peroxissomais, que poderão ser usados com alvos terapêuticos na infeção viral.
Programa Doutoral em Biomedicina

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
98
Background: Milk-fat globule EGF factor 8 (MFG-E8) is a secreted glycoprotein found in milk-fat globule derived from lactating mammary. MFG-E8 proteins express mainly in the mammary cells and immuno-modulating cells such as tingible-body macrophages in spleen and lymph node. MFG-E8 contains EGF domains (Epidermal growth factor-like) and C1/C2 domains (Coagulation factor V and VIII type C-like) for integrin αvβ3/5 on (MΦ) binding and phosphatidylserine (PS)(on apoptotic cell) recognition, respectively. The importance of MFG-E8 function as a bridging molecule during apoptotic cell clearance has been emphasized in recent years. Decreased phagocytic clearance and accumulation of apoptotic cells was noted in MFG-E8-deficient mice. Intriguingly, the 40-week-old female MFG-E8-deficient mice present higher titer of autoantibodies and suffered splenomegaly/glomerulonephritis more prevalently than the male mice, which were characterized in human systemic lupus erythematosus (SLE). These results indicate the correlation between MFG-E8 deficiency-related apoptotic cell clearance defect and SLE pathogenesis. Our previous studies revealed that the variant on MFG-E8-76th residue (76Met) was a predisposing factor for human SLE. Interestingly, a concensus Kozac sequence (A/G-3NNATGG+4) resides around residue 76Met, which may potentially act as an alternative translation initiation site and subsequently produce short form MFG-E8-Δ1-75 in cells of MFG-E8-76Met variant type. Methods: Several approaches were applied to test the hypothesis whether there is alternative translation - 1. Molecular cloning of MFG-E8 cDNA and express recombinant MFG-E8 protein in E. coli. 2. Examine whether MFG-E8-76Met variant form perform alternative translation in different MFG-E8-76 variant forms-transfected 293T cell lines and PBMC-derived human macrophages with different MFG-E8-76 variant forms. 3. Detect serum MFG-E8 expression level in SLE cohort to realize the correlation between MFG-E8-76 variations and serum MFG-E8 level. Results: 1. MFG-E8 cDNA was successfully cloned and then expressed in E. coli. However, the protein expression levels are considered low even after optimizing the protein induction conditions. Further protein expression was hampered due to low expression level. 2. Short form MFG-E8 protein was produced in MFG-E8 (Δ1-75)-transfected 293T cell lines but the expression efficiency were considerably lower than that found in 293T cell transfected with full length MFG-E8. 3. No short form MFG-E8 protein was observed in MFG-E8-76Met-transfected 293T cell lines. 4. No short form MFG-E8 protein were observed in peripheral blood monocytic cells-derived macrophage of MFG-E8-76Met variant form. Interestingly, the MFG-E8 detected in human macrophage was of smaller size than the expected. The protein expression level differed independently of MFG-E8-76 variant forms. 5. Serum MFG-E8 expression levels in SLE cohort were significantly higher than that of non-lupus controls but the elevated MFG-E8 level did not show significant correlation with MFG-E8-76 variant forms. Conclusions: 1. MFG-E8-76th codon ATG does not show to be an appropriate alternative translational start site. 2. Serum MFG-E8 levels were higher in part of SLE patients than in healthy controls but the elevated MFG-E8 level did not correlation with the MFG-E8-76 variant forms. 3. MFG-E8 expression levels had no correlation with the MFG-E8-76 variant forms in human macrophage. The proteins determined in human macrophage were smaller than estimated size suggested that there have distinct processes in human macrophage. The differential processing and expression of MFG-E8 in the phagocytes await further investigation.