Abstract Type XIII collagen is an integral membrane protein in type II orientation. In cells and tissues type XIII collagen has been located in various adhesive structures, like focal adhesions. Due to this, its biological role has been implicated in cell adhesion. This collagen also exists as a soluble protein due to the release of the ectodomain from the plasma membrane. In this thesis, ectodomain shedding, i.e. enzymatic release of the extracellular domain, was studied in detail, focusing on the phenomenon as it occurs in mammalian cells. It was found that the ectodomain is released by members of the mammalian proprotein convertase family, e.g. furin. Shedding was shown to take place at the cell surface, but based on additional observations, this cleavage may also take place intracellularly in the Golgi apparatus. Various intracellular mechanisms, depending on cell type, were found to be involved in the regulation of ectodomain shedding. Apparently, due to the liberation of the ectodomain, the level of type XIII collagen on the plasma membrane is maintained at a relatively even amount. The released ectodomain was shown to retain biological activity. It showed distinct matrix-specificity so that on vitronectin its influence on cell functions was anti-adhesive, anti-migratory, anti-proliferative and non-supportive of cell spreading. It was also demonstrated to affect the fibronectin matrix assembly in a manner that resulted in reduced amounts of the fibrillar fibronectin matrix. A large collection of human epithelial and mesenchymal cancer samples were screened for type XIII collagen mRNA expression and compared to the expression levels of pre-malignant and normal samples. It was discovered that malignant transformation upregulates the expression of type XIII collagen in mesenchymal cancers and particularly in the stroma of epithelial cancers, more so than in cancer epithelia. TGF-β1 was demonstrated as one factor contributing to the stimulation of expression. Based on cell culture experiments in this study, it was also deduced that the upregulated expression of type XIII collagen and the concomitant shedding of the ectodomain can remodel the tumour stroma, making it inauspicious for adhesion-dependent cell functions, particularly in vitronectin-rich milieu.

<p>The transforming growth factor beta (TGF-beta) signaling pathway is a critical regulator of multiple biological processes that are involved in cancer progression, such as proliferation, migration, invasion and metastasis. TGF-beta ligands bind to multiple high-affinity receptors (TbetaRI, TbetaRII, TbetaRIII), whose expression on the cell surface, and subsequent ability to transduce signaling, can be modulated by ectodomain shedding. </p><p> TbetaRIII, also known as betaglycan, is the most abundantly expressed TGF-beta receptor. TbetaRIII suppresses breast cancer progression through inhibiting migration, invasion, metastasis, and angiogenesis. TbetaRIII binds TGF-beta ligands, with membrane-bound TbetaRIII presenting ligand to enhance TGF-beta signaling. However, TbetaRIII can also undergo ectodomain shedding, releasing soluble TbetaRIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound TbetaRIII on TGF-beta signaling and breast cancer biology, here I describe TbetaRIII mutants with impaired (Delta-Shed-TbetaRIII) or enhanced ectodomain shedding (SS-TbetaRIII). Relative to wild-type (WT)-TbetaRIII, Delta-Shed-TbetaRIII increased TGF-beta signaling and blocked TbetaRIII's ability to inhibit breast cancer cell migration and invasion. Conversely, SS-TbetaRIII, which increased soluble TbetaRIII production, decreased TGF-beta signaling and increased TbetaRIII-mediated inhibition of breast cancer cell migration and invasion. </p><p> TbetaRI is released from the cell surface by a common sheddase of the A disintegrin and metalloproteinase (ADAM) family, ADAM17. This shedding event results in a downregulation of TGF-beta signaling. Here, I present evidence that a closely related protease, ADAM10, may be a novel sheddase for TbetaRI. A specific ADAM10 inhibitor was able to increase cell surface expression of TbetaRI, and decrease levels of circulating soluble TbetaRI in vivo. Interestingly, inhibition of ADAM10 concurrently increased shedding of TbetaRIII, and was able to alter TGF-beta signaling in a TbetaRIII-dependent manner. </p><p> </p><p> Together, these studies suggest that ectodomain shedding of TGF-beta receptors is an important determinant for regulation of TGF-beta-mediated signaling and biology.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20667号 / 医博第4277号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 妹尾 浩, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18682号 / 医博第3954号 / 新制||医||1007(附属図書館) / 31615 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 野田 亮, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Aujourd’hui, le Virus de l'Hépatite C (VHC) infecte plus 70 millions de personnes dans le monde. L’Organisation mondiale de la santé prévoit l’élimination du virus VHC d’ici 2030, grâce aux récentes découvertes dans le milieu du développement médical. Ces derniers ont conduit à la production des antiviraux pangenotypiques à action directe (ADD). Le VHC est un virus enveloppé de l’ARN, avec une polarité positive. Il est constitué de nucléocapside entouré d’une membrane lipidique. La nucléocapside contient l’acide ribonucléique (ARN) et la protéine core. La membrane lipidique quant à elle contient à la surface les glycoprotéines E1 et E2. Ainsi ces protéines, sont les premières à rencontrer les hépatocytes, c’est donc grâce à elles que le virus parvient à entrer dans les cellules. Parmi les deux protéines, l’E2 a été la mieux caractérisée pour ses fonctions de liaisons aux récepteurs spécifiques. De plus les anticorps neutralisants ciblent majoritairement cette protéine. En se basant sur le fait que ce virus est membre de la famille des Flaviviridae, il a été suggéré par analogie, que le VHC contient des protéines de fusion de classe II et que la protéine E2 est la protéine de fusion. Cependant, les structures cristallines récentes d’E2 ont révélé qu'il lui manquait les caractéristiques structurelles des protéines de fusion de classe II. Ainsi, tous les regards se sont tournés sur la glycoprotéine E1, suggérant qu’elle est responsable de l’étape de fusion, seule ou à l’aide d’E2. En effet, la partie N-terminale de l'ectodomaine E1 a été récemment cristallisée. La caractérisation des résidus conservés dans cette région a démontré son importance pour l'infectivité du virus, pour l'interaction entre E1 et E2, ainsi que pour son implication dans l'interaction avec les récepteurs du VHC. En soutenant le rôle potentiel d'E1 dans le processus de fusion, différents segments de l'extrémité C-terminale de l'ectodomaine seraient impliqués dans les interactions avec les membranes modèles. Nous avons étudié en particulier deux régions d’intérêt. La première située dans la zone du peptide de fusion putatif (PFP) entre les acides aminés 270 et 291. Cette région se compose des séquences hydrophobes, soutenant son implication dans l'étape de fusion. La deuxième région englobant les acides aminés 314-342, d’une activité membranotrope située à proximité de la zone transmembranaire d’E1, a été démontrée par la cristallographie aux rayons X et les études de RMN comme comprenant deux hélices α (α2 et α3).Nous avons introduit 22 mutations dans la partie C-terminale de l'ectodomaine E1 dans le contexte d'un clone infectieux JFH1. Nous avons remplacé les résidus les plus conservés par de l'alanine, puis analysé l'effet des mutations sur le cycle de vie du virus. Vingt des vingt-deux mutants ont été atténué ou ont perdu leur pouvoir infectieux, ce qui indique leur importance dans le cycle viral. Nous avons observé différents phénotypes; certaines mutations ont modulé la dépendance du virus vis-à-vis des récepteurs CLDN1 et SRBI pour l’entrée cellulaire. Plusieurs mutations dans la région PFP, ont affecté la sécrétion et l'assemblage du virus, ainsi que l'hétérodimérisation E1E2. D’autres mutations, telles que les mutations de l'hélice α2 ont entraîné une atténuation grave ou une perte complète d'infectivité, sans affecter le repliement d’E1 et E2, ni la morphogenèse virale. Une caractérisation plus poussée de certains mutants au sein de la région hélice α2 a suggéré l'implication de cette région dans une étape tardive de l'entrée du VHC. Enfin, nos résultats montrent le rôle important joué par la glycoprotéine E1 dans l'hétérodimérisation de E1E2, la morphogenèse du virus, ainsi que son interaction avec les récepteurs du VHC et son implication potentielle dans l'étape de fusion. / Hepatitis C virus is currently estimated to infect around 71 million people around the world. However, recent advances in drug development led to the generation of pangenotypic direct acting antivirals (DAA), which may make it possible to eliminate HCV by 2030 as planned by the World health organization (WHO). HCV is a small RNA enveloped virus of positive sense. The RNA is encapsidated and surrounded by a lipid bilayer in which the E1 and E2 envelope glycoproteins are anchored on the surface. Thus, E1 and E2 are the first viral proteins to encounter the hepatocytes and mediate the entry step. HCV entry into hepatocytes is a sophisticated process that includes several steps ranging from interaction of glycoproteins with cellular host attachment factors and HCV specific-receptors, which is followed by internalization via clathrin-mediated endocytosis. Finally, viral and endosomal membranes merge at acidic pH leading to the release of viral RNA into the cytoplasm. Among the two glycoproteins, E2 has been the better characterized, as it is responsible for binding to cellular receptors and targeted by neutralizing antibodies. As a member of the Flaviviridae family, it has been suggested by analogy that HCV encodes class II fusion proteins and that E2 is the fusion protein. Nevertheless, the recent crystal structures of E2 revealed that it lacks structural features of class II fusion proteins. Thus, E1 glycoprotein became under the spotlight with the assumption that it is responsible for the fusion step whether alone or with the help of E2. Indeed, the N-terminal part of E1 ectodomain was recently crystallized, and the characterization of conserved residues within this region demonstrated its importance for virus infectivity, E1E2 interaction as well as its involvement in the interplay with HCV receptors. Supporting the potential role of E1 in the fusion process, different segments in the C-terminal of the ectodomain have been reported to be involved in interactions with model membranes. In particular, we investigated two regions of interest. The first one located in the putative fusion peptide (PFP) region between amino acid 270 and 291, containing hydrophobic sequences, supporting its involvement in the fusion step. The second region spanning amino acids 314-342, a membranotropic region located proximal to the transmembrane region of E1 and has been shown by X-ray crystallography and NMR-studies to comprise two α-helices (α2 and α3). We introduced 22 mutations in the C-terminal part of E1 ectodomain in the context of a JFH1 infectious clone. We replaced the most conserved residues with alanine and analyzed the effect of the mutations on the viral life cycle. Twenty out of the 22 mutants were either attenuated or lost their infectivity, indicating their importance for the viral life cycle. We observed different phenotypes; some mutations modulated the dependence of the virus on CLDN1 and SRBI receptors for cellular entry. Most mutations in the PFP region affected virus secretion and assembly as well as E1E2 heterodimerization. Nevertheless, the majority of mutations in the α2-helix (aa 315-324) led to severe attenuation or complete loss of infectivity without affecting E1E2 folding or viral morphogenesis. Further characterization of some mutants within this region suggested the involvement of the α2-helix in a late step of HCV entry. Finally, our results show the important role of E1 played in E1E2 heterodimerization, virus morphogenesis, interaction with HCV receptors and its potential involvement in the fusion step.

Das Hüllglykoprotein Hämagglutinin (HA) von Influenzavirus ist verantwortlich sowohl für die Bindung als auch für die nachfolgende Fusion der viralen Hülle mit der endosomalen Membran. Eine Analyse der 3D Struktur der HA-Ektodomaine zeigt, dass die Stabilität des Proteins sowohl durch kovalente als auch durch nicht-kovalente Wechselwirkungen bedingt ist. Die Konformationsänderung von HA bei saurem pH-Wert weißt auf eine mögliche Rolle von Protonierungseffekten auf ionisierbare Aminosäuren hin. Untersuchungen zur Bedeutung geladener Aminosäuren und Salzbrücken für die Struktur des HA wurden auf der Grundlage von ‚site directed mutagenesis’ durchgeführt. Der Einfluss der Mutationen auf die Konformationsänderung und die Fusionsaktivität von HA wurden durch einen Proteinase K-Assay bzw. Fluoreszenzmikroskopie erfasst. Die Ergebnisse beider Methoden wurden miteinander korreliert. Abgesehen von der Mutante R109E zeigten Wildtyp-HA und alle anderen Mutanten eine vergleichbare Oberflächenexpression. Die beobachteten Unterschiede in der pH-Abhängigkeit der Konformationumwandlung zwischen Wildtyp-HA und HA-Mutanten zeigen, daß eine Zerstörung von Salzbrücken und ggf. eine Erhöhung der elektrostatischen Abstoßung an den betrachteten Kontakstellen sehr wahrscheinlich eine Herabsetzung der energetischen Barriere der Konformationsumwandlung verursacht. Dieser Ergebnisse erklären die molekularen Grundlagen des erhöhten pH-Schwellwertes der HA-Konformationsumwandlung von Amantadin-resistenten Influenzaviren. Im Gegensatz wurde für Mutanten, die die Stabilität von HA erhöhten, keine Konformationsumwandlung bei einem pH-Wert beobachtet, der typisch für die Konformationumwandlung von Wildtyp-HA war. Aminosäuren, die denen dieser stabiliserenden Mutationen entsprachen, wurden in einer natürlichen Influenzavirusvariante – A/JPN/305/57 – gefunden. Die Bedeutung von Ladungen für die Stabilität der HA-Ektodomaine wird dadurch unterstrichen, dass eine Konservierung einer positiven Ladung und insbesondere eines Argininrestes in der Position 109 (Nummerierung auf der Basis von HA X31) für alle Influenzaviren A und B gefunden wurde. Die Ergebnisse der Arbeit zeigen, dass sehr wahrscheinlich eine komplexe Salzbrücke an der Kontaktfläche zwischen HA1 und HA2 für alle Influenzaviren A evolutionär konserviert ist. / Hemagglutinin (HA), a major envelope glycoprotein is responsible for fusing viral and endosomal membranes during influenza virus entry. The analysis of 3D crystal structure of the HA ectodomain shows that the stability of protein is maintained by both non-covalent and covalent interactions. The conformational change of HA at low pH indicates a role for protonation effects of the ionisable amino acids. Structural investigations were done using “site directed mutagenesis” in order to conceive the importance of charged amino acids and more emphatically the involvement of salt bridges. The effect of mutations on the conformational change and fusion activity was probed by proteinase K assay and fluorescence microscopy respectively. It was observed that HA-wt and all the mutants except R109E showed comparable surface expression. The difference in pH threshold between the HA-wt and the mutants showed that breakage of salt bridge and further incorporation of repulsion at the considered interfaces would lower the energy barrier requirements for the conformational change. The results explain the molecular basis of the higher pH threshold for naturally occurring amantadine resistant mutants. On the other hand, mutants designed to stabilise the HA were resistant to conformational changes at those pH values which typically trigger the conformational change of HA-wt. Coincidentally these mutations were found to be existing in the natural variant of H2 Japan subtype (A/JPN/305/57). Interestingly, the study shows that a positive charge and, more specifically, an Arg residue at position 109 (numbering based on X-31 strain) is conserved in all of the influenza A and B viruses underlining the relevance of electrostatic interactions for the HA stability. Aptly a complex salt bridge at the interface of HA1 and HA2 is probably conserved evolutionarily in all the members of influenza A virus.

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 &Aring; resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

An injured vascular system has a substantial impact on an individuals overall health, and an understanding of the mechanisms that underlie blood vessel pathophysiology is required for the development of rational and effective treatment strategies. The phenotypic modulation of smooth muscle cells (SMC) during vascular injury, characterized by altered adhesion, migration and synthetic behavior, plays an important role in the eventual outcome. Specifically, the ability of SMCs to adhere to and remodel their extracellular environment via regulation of the syndecan class of cell adhesion molecules dictates the response of the vascular wall to local injury. The effect of in vitro syndecan-4 regulation on SMC adhesion was investigated through the use of a glass microsphere centrifugation assay, and an antisense-mediated reduction in gene expression was found to correlate with decreased adhesive strength. Regulation of syndecan-1, syndecan-2, and syndecan-4 gene expression was observed experimentally by mechanical strain of SMCs. Using real-time polymerase chain reaction (PCR), the kinetics of both static and cyclic mechanical strain were found to modify the gene expression in a time and strain magnitude-dependent manner unique to each syndecan. In particular, the responses of syndecan-4 were acute, but transient, while the evolution of syndecan-1 and syndecan-2 regulation was delayed by comparison. Mechanical strain also modulated syndecan-4 protein expression and ectodomain shedding, as measured by Western immunoblotting, and this effect was found, through selective inhibition, to be at least in part dependent on mitogen-activated protein (MAP) kinase signaling. In particular, intact extracellular signal-regulated MAP kinase (ERK) 1/2 and c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK/SAPK) signaling pathways were found to be required for the observed strain-induced shedding. These findings offer a better understanding of syndecan function in response to mechanical strain and suggest potential new mechanisms by which physical forces may modulate vascular SMC behavior and regulation during normal physiology, pathologic conditions, and engineered arterial substitute development.