Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 69-82).

Oι ιντεγκρίνες είναι κυτταρικοί υποδοχείς, οι οποίοι αλληλεπιδρούν με την εξωκυττάρια ύλη. Μέχρι σήμερα έχουν ανακαλυφθεί 18α και 8β υπομονάδες συνδυασμός των οποίων δημιουργεί 24 διαφορετικές ιντεγκρίνες . Ο ρόλος τους είναι η σύνδεση της εξωκυττάριας με την ενδοκυττάρια ύλη, καθώς ενεργοποιούν ενδοκυττάρια σηματοδοτικά μονοπάτια που εμπλέκονται στην επιβίωση, μετανάστευση, πολλαπλασιασμό και απόπτωση των κυττάρων, λειτουργίες ζωτικής σημασίας για τον οργανισμό. Ιδιαίτερο ενδιαφέρον παρουσιάζει η α4β1 ή VLA-4 ιντεγκρίνη, η οποία αποτελείται από μία α4 (155 kDa) και μία β1 (150 kDa) υπομονάδα. Εκφράζεται στα κύτταρα του μυελού των οστών εκτός από τα ουδετερόφιλα και έχει δύο κύριους φυσικούς προσδέτες, το αγγειακό μόριο κυτταρικής προσκόλλησης-1 (VCAM-1) και την φιμπρονεκτίνη. Ο ρόλος της α4β1 ιντεγκρίνης είναι ζωτικός στις φλεγμονώδεις διαταραχές ενώ φαίνεται να εμπλέκεται και στην αγγειογένεση. Στόχος της παρούσας διατριβής είναι η ανάπτυξη νέων α4β1 κυκλικών πεπτιδικών προσδετών και η μελέτη της δράσης αυτών ως αναστολείς της φλεγμονής και της αγγειογένεσης. Τα συντεθέμενα ανάλογα έχουν μία βασική κυκλική δομή, ενώ τροποποιήσεις έχουν πραγματοποιηθεί τόσο στην Ν-τελική όσο και στην C-τελική αλληλουχία. Η σύνθεση των αναλόγων πραγματοποιήθηκε με την Fmoc/But μεθοδολογία επί στερεάς φάσεως, χρησιμοποιώντας ως στερεό υπόστρωμα τις ρητίνες 2-χλωροτρίτυλο και την Rink Amide MBHA, για την παραλαβή C-τελικού καρβοξυλίου ή αμιδίου αντίστοιχα. Η βασική κυκλική δομή επιτεύχθηκε μέσω σχηματισμού δισουλφιδικής γέγυρας χρησιμοποιώντας ως οξειδωτικό μέσο διμεθυλοσουλφοξείδιο (DMSO) είτε σε υγρή, είτε σε στερεά φάση. Στην παρούσα φάση, πραγματοποιείται μελέτη των συντεθέντων αναλόγων in vivo στη χοριοαλλαντοϊκή μεμβράνη του εμβρύου όρνιθας (CAM) ως αναστολείς της αγγειογένεσης, ενώ βιολογικά πειράματα θα διεξαχθούν για την πιθανή χρήση αυτών ως αντιφλεγμονώδεις παράγοντες. Επιπρόσθετα, μελετάται η διαμόρφωση των παραπάνω πεπτιδικών αναλόγων, μέσω τεχνικών NMR φασματοσκοπίας και μοριακής μοντελοποίησης. / Integrins are cell surface receptors, which interact with the extracellular matrix. They are heterodimers consisting of α and β subunits. Until now, 18 α subunits and 8 β subunits have been discovered that form 24 different integrins. Their role is the connection of the extracellular matrix with the intracellular cytoskeleton, as they activate intracellular signaling pathways regulating the migration, proliferation, survival and apoptosis of the cells, functions of vital importance for the organism. An integrin with particular interest is α4β1 or VLA-4 integrin, which consists of a α4 (155 kDa) and a β1 (150 kDa) subunit. It is expressed on bone marrow derived cells, except on neutrophils, and has two main natural ligands, fibronectin and vascular cell adhesion molecule-1 (VCAM-1). The role of α4β1 is vital for the inflammation process, while it also seems to participate in tumor angiogenesis. The aim of this research is the development of α4β1 cyclic peptide antagonists and their study as inflammation and tumor angiogenesis inhibitors. The above analogues have a basic cyclic peptide structure, while modifications have been achieved at the N-terminus and C-terminus sequences. The analogues were synthesised by Fmoc/But solid phase methodology utilizing Rink Amide MBHA and 2-chlorotrityl-chloride resin to provide C-terminal amide and carboxylic acid, respectively. The basic cyclic unit of the above analogues was achieved through the formation of a disulfide bridge, using as oxidant dimethylsulfoxide (DMSO), in either solution or solid phase methodology. At present, the above analogues are tested in vivo in chick embryo chorioallantoic membrane (CAM) model as anti-angiogenic agents, while biological experiments will be performed for their potential use as anti-inflammatory agents. Furthermore, the conformation of the above peptide analogues is studied in solution environment, by NMR spectroscopy techniques and molecular modeling.

Integrinas são glicoproteínas transmembrânicas envolvidas na adesão célula-célula e célula-proteina da matriz extracellular (ECM) que participam da migração e fusão de células trofoblásticas gigantes (TGC) com células do epitélio materno no placentoma bovino e interagem com inibidores teciduais de metalloproteinases (TIMPs), considerados importantes reguladores das metaloproteinases de matriz (MMPs), que são reconhecidas como passo limitande para ação de enzimas que atuam no remodelamento da ECM na implantação e na gestação. Uma vez que as integrinas são consideradas responsáveis pela manutenção da arquitetura tecidual e que as MMPs podem regular a degradação e a reconstituição da ECM necessária para a invasão do trofoblasto, temos como objetivo verificar uma possível relação das integrinas com alterações placentárias comumente observadas em gestações de animais clonados (SCNT). Para avaliar a funcionalidade placentária, a expressão do lactogênio placentário (PL) e do antígeno Ki-67 foram também verificadas. Placentomas bovinos foram coletados e divididos em 3 grupos: 1) início (n = 6) e 2) final (n = 3) da gestação de animais derivados de monta natural (n=9) e final da gestação de animais clonados (n=8), clones machos (n=4) e clones fêmeas (n=4). As proteínas foram localizadas por imuno-histoquímica indireta, exceto as subunidades de integrina &alpha;6, &beta;1, &alpha;V e &beta;3 que foram localizadas por imunofluorescência. A especificidade dos anticorpos e expressão protéica foi confirmada por Western blot e a expressão do RNAm foi avaliada por meio de RT-PCR e PCR em tempo real quantitativo. As células positivas para a laminina, TIMP-2, Ki-67 e lactogênio placentário (PL) foram quantificadas e para comparação entre os grupos. A proteína das subunidades &alpha;6 do receptor de integrina e &beta;1 foi observada nos animais clonados e não clonados nos estromas materno e fetal e porção basal dos epitélios materno e fetal e foi co-localizada com a laminina. A proteína das subunidades &alpha;V e &beta;3 do receptor de integrina foi observado nos estromas materno e fetal e foi co-localizado com a fibronectina. O TIMP-2 foi exclusivamente e especificamente localizado nas TGCs no início e final da gestação de animais clonados e não clonados, mas não houve diferença significativa em sua expressão. O PL apresentou a mesma localização do TIMP-2 em todos os grupos analisados. Houve diferença significativa (p<0,05) entre 270d e clones ao se comprar a expressão relativa do RNAm da subunidade de integrina &beta;1 e do lactogênio placentário e ao se comparar a expressão protéica por western blot da laminina e do TIMP-2. Foi possível observar diferença significativa (p<0,05) entre o grupo controle e o grupo de animais clonados ao se quantificar o número de TGCs positivas para a laminina e para o lactogênio placentário. Apesar de algumas diferenças terem sido observadas na expressão de integrinas e de proteínas da ECM, sugere-se que a interação das integrinas com seus ligantes da ECM a termo não é um determinante da sobrevivência neonatal de bovinos clonados. Contudo, outros estudos são necessários para esclarecer se estas proteínas representam elementos chave na placentação de bovinos clonados em início de gestação. As diferenças observadas na expressão de integrinas e principalmente do PL entre clones fêmeas e machos sugerem possível influência de genes associados ao cromossomo sexual ou imprinting. / Integrins are transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix (ECM) adhesion and signal transduction. They participate in the migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells in bovine placentomes, and interact with tissue inhibitors of metalloproteinases (TIMPs), considered important regulators of matrix metalloproteinases (MMPs). MMPs are rate-limiting enzymes degrading ECM proteins during tissue remodeling around embryo implantation, pregnancy and parturition. Since integrins are considered to be responsible for the maintenance of the architecture within tissues and MMPs may also regulate degradation and reconstitution of ECM required for trophoblast invasion, we aimed to verify a potential relation of integrins with common placental alterations observed in cloned animals (SCNT). To verify the placental functionality, expression of placental lactogen (PL) and Ki-67 antigen was also assessed. Bovine placentomes were collected and divided into 3 groups: 1) early and 2) late gestation derived from natural mating (n=9) and 3) late gestational bovine clones (n=8), also divided into male clones (n=4) and female clones (n=4). All proteins were localized by indirect immunohistochemistry except for integrin subunits &alpha;6, &beta;1, &alpha;V and &beta;3 that were shown by immunofluorescence. The antibody specificity and protein expression were confirmed by Western blot. Expression of mRNA was evaluated using RT-PCR and quantitative Real Time PCR. Positive cells for laminin, TIMP-2, Ki-67 and placental lactogen were quantified for comparisons among groups. The protein of integrin receptor subunits &alpha;6 and &beta;1 was observed in both cloned and non-cloned animals in the fetal and maternal stroma and at the basal membrane of maternal and fetal epithelial cells, co-localized with laminin and with collagen IV. The integrin receptor subunits &alpha;V and &beta;3 were observed in the fetal and maternal stroma, co-localized with fibronectin. TIMP-2 was specifically and exclusively localized in TGC in the early and term gestation in both cloned and non-cloned animals. PL has shown the same localization of TIMP-2 in all analyzed samples. Statistically significant differences (p<0,05) between term gestation of non-cloned and cloned animals were observed comparing the mRNA expression of integrin &beta;1 subunit and placental lactogen and the protein expression of laminin and TIMP-2. There were also statistically significant differences (p<0,05) between non-cloned and cloned animals in the number of laminin and placental lactogen positive cells. Despite some differences in integrin and ECM proteins expression, our results suggest that the interaction between integrins and their ECM ligands in term gestation is not a determinant for the survival rate of newborn cloned calves. However further studies are necessary to elucidate if integrins represent key elements in the early placentation of SCNT bovines. The differences found in the integrin and principally in the placental lactogen expression between female and male cloned calves suggests the influence of sex-chromosome associated genes or imprinting.

Abstract Collagen IX belongs to the superfamily of collagenous proteins and is present on the surface of the heterotypic collagen fibrils that are predominantly composed of collagen II, and also collagen XI. The major sites of expression of collagen IX include the articular cartilage, intervertebral disc, inner ear and the vitreous body of the eye. Previous reports have indicated that mutations in the genes encoding the three polypeptide chains of collagen IX may lead to intervertebral disc disease and multiple epiphyseal dysplasia, a chondrodysplasia characterized by early onset osteoarthritis. These observations and results from genetically modified mouse lines suggest that collagen IX is crucial in the maintenance of the long-term integrity of tissues. However, the structure-function relationship as well as detailed information concerning the functional roles of this protein has remained unclear. Recombinant human collagen IX was obtained using an insect cell expression system. Besides full-length molecules, five truncated variants of collagen IX were produced to examine chain association and trimerization. Contrary to previous observations, it was shown that the COL1 and NC1 domains are not essential for trimerization. Instead, they seem to play an important role in the specificity of chain selection. The results also suggest that the N-terminal domains, NC3 or COL3, are required for complete folding and stabilization of collagen IX molecules, implicating cooperativity between different domains in the folding process. Collagen IX was found to mediate cell adhesion and bind efficiently to collagen receptor integrins α1β1, α2β1, α10β1 and α11β1. The binding was found to represent a novel type of mechanism, and the binding site of the integrin I domain was located at the N-terminal end of the COL3 domain in collagen IX. The obtained results suggest that the FACITs may play an important role as mediators of cell adhesion to collagen fibrils. Antibodies binding to human recombinant collagen IX were measured among 53 patients with seropositive rheumatoid arthritis (RA). These autoantibodies were significantly elevated among the RA patients when compared to the controls, suggesting that autoantibodies to collagen IX show diagnostic potential in early RA. However, no association was found between the antibody levels and outcome.

Proteins represent major constituent of the extracellular matrix which plays an important role in the formation, maintenance and remodeling of tissues, this project focuses on adsorption of two specific serum proteins fibronectin (FN) and vitronectin (VTN) responsible for mediating cell matrix interaction through integrin binding, tripeptide Arg-Gly-Asp (RGD) sequence found in these protein features are recognized by αβV3 integrin which ultimately helps in clot formation.

Large efforts have been made to target integrin αVβ3 of endothelial cells. We have successfully developed a new class of protein (Ref to as ProAgio) by rational protein design using a stable host protein, domain 1 of cell adhesion protein CD2. ProAgio is designed to target integrin αVβ3 at a novel site and induces angiogenic endothelial cell apoptosis by recruiting and activating caspase 8 to the cytoplasmic domain of the targeted integrins. Tests with tumor xenograft models show that ProAgio strongly inhibits tumor growth. Histology analyses indicate that tumor vessels are reduced, while the established vasculatures are not affected. Toxicity analyses demonstrate that ProAgio is not toxic to mouse. Our study develops an effective anti-angiogenesis agent and provides a new platform for development of therapeutics by targeting integrins. We have successfully developed an anti-angiogenesis protein targeting integrin αVβ3 at a novel site by rational protein design. The developed agent is not toxic to non-cancerous blood vessels and other tissue/organs, providing an excellent candidate for future potential clinical development. Our developed protein is one of the very few examples that do not act through targeting VEGF/VEGFR or any other RTK pathways. The βA groove is present in almost all other β integrins. This approach may be applicable to develop agents targeting the similar βA groove of other integrin pairs, which can address wide array of pathological conditions such as AMD, Rheumatoid Arthritis, Osteoporosis etc.

The niche in which stem cells reside is involved in the regulation of stem cell fate, such as differentiation and self-renewal, by providing ECM proteins, growth factors, cell-cell interactions and balancing chemical factors such as the level of oxygen and pH. ECM proteins are involved in maintaining stemness of stem cells and in regulating differentiation via integrin-mediated signalling. Following the interaction of ECM proteins with integrins, integrins cluster and interact with large complexes of signalling proteins. These adhesion complexes have been reported to contain at least 150 proteins, which have been termed the adhesome. Adhesion complex proteins interact with the actin cytoskeleton and signalling pathways to play an essential role in stem cell fate. The hypothesis in this study was that the interaction of stem cells via integrin receptors with ECM proteins, lead to changes in the abundance or composition of adhesion complexes, which potentially activates signalling pathways involved in either maintaining or differentiation of stem cells. In this study, three principal advances have been made:First, a method was developed using ligand-coated magnetic beads for the isolation of integrin-associated complexes from pluripotent human embryonic stem cells (hESCs). The isolated integrin-associated complexes from hESCs were analysed by proteomic methods, which led to the detection of key integrin-associated adhesion proteins such as talin, vinculin, alpha actinin 4, filamin B, filamin C and zyxin. Second, isolation of integrin-associated complexes from multipotent MSCs was performed using a method based on “de-roofing” MSCs from FN or PDL coated plastic dishes, leading to the detection of key adhesome components by mass spectrometry. Ontological analysis of proteins enriched on FN demonstrated the enrichment of adhesion complexes. Third, following the induction of multipotent MSCs into early adipogenic MSCs and the isolation of integrin-associated complexes from early adipogenic MSCs and undifferentiated MSCs, core adhesome components were identified in induced and non-induced MSCs, with induction hypothesised to cause putative changes in the FN-induced adhesome network. Also, the level of adhesion complexes increased significantly in MSCs on FN upon induction into adipocytes compared to non-induced MSCs on FN and versus the control as shown by bioinformatics analysis. This data led to the hypothesis that upon induction of MSCs into adipocytes the abundance of proteins in integrin-associated complexes or the number of adhesion complexes increases.In conclusion, in this study two biochemical affinity methods were developed for the isolation of integrin-associated complexes from hESCs and MSCs, using ligand coated magnetic beads and ligand-coated plastic dishes. The development of these methods led to the isolation of adhesion-related proteins from pluripotent hESCs and differentiated MSCs and the detection of a pattern of changes in the abundance of adhesion related proteins in differentiated MSCs incubated on FN. The development of methods for the isolation of adhesion related complexes from stem cells can lead to a better understanding of the role of adhesion in differentiation and maintenance of pluripotency in stem cells. A better understanding of adhesion could have future implications in obtaining pure populations of undifferentiated stem cells for cell-based therapies and differentiated cells for the use in tissue engineering and repair.

Acute myocardial infarction (MI) is the end result of many cardiovascular diseases and is one of the leading causes of death in the western world. Cell therapy, using circulating angiogenic cells (CACs) or CD34+ cells from peripheral blood, is one approach under investigation for restoring blood flow and function to the ischemic heart. However, the numbers of CACs and CD34+ circulating cells are inversely proportional to the severity of cardiovascular disease and age; therefore, there is a need to increase their numbers and/or function for therapy. One possibility is to enhance the therapeutic potential of the cells with the use of a biomaterial. In this study, we used a collagen matrix to culture human CD34+ circulating cells, and evaluated the effect of the matrix on CD34+ cell properties and function. The matrix was able to successfully increase proliferation, migration, CD34+ phenotype and branching in an angiogenesis assay. These functional benefits may be associated with the sonic hedgehog (Shh) pathway. The collagen matrix was previously shown to enhance the function of healthy CACs, but its ability to do the same for CACs from coronary artery disease patients is unknown. In this study, the matrix was shown to enhance the viability, proliferation and angiogenic potential of patient CACs. Furthermore, gene expression for integrins and Shh pathway components in the sub-population of CD34+ cells was similar between patient and healthy donors when isolated from CACs. This work provides insight into the mechanisms for the observed matrix-enhanced function of therapeutic CACs and CD34+ cells from both healthy and CAD patient donors.

Cell adhesion to the extracellular matrix is mediated by the integrin family of adhesion receptors. Integrin receptor engagement initiates the formation of multimolecular protein complexes, termed integrin adhesion complexes (IACs), at the cell membrane. IACs are complex signalling hubs that are enriched in tyrosine-based phosphorylation events and form a mechanochemical connection between integrin receptors and the actin cytoskeleton. Dysregulation of individual IAC components has been reported to influence a wide range of biological processes that contribute to disease. Literature-curated and proteomic analyses of IACs have revealed an unanticipated molecular complexity of IACs in a variety of experimental contexts; however, a global consensus view of the composition of IACs, and a description of how the complex network of interactions in IACs influences global cell function, is currently lacking. Here, multiple existing and new proteomic datasets detailing the protein composition of IACs were analysed to identify a systems-level description of IACs and to enable interrogation of IAC structure, topology and dynamics. Quantitative IAC proteomes derived from multiple cell types were integrated to generate a 2,412-protein ‘meta-adhesome’ database of proteins enriched to fibronectin-induced IACs. To investigate the putative functional adhesion landscape in an objective manner, the meta-adhesome was analysed using a combination of hierarchical clustering, gene ontology and interaction network analyses. An emergent property of the meta-adhesome was the definition of a consensus adhesome: 60 proteins commonly identified from IAC datasets that likely represent an IAC protein core composition. The consensus adhesome highlights how integrins connect to actin via multiple pathways and consists of both canonical and underappreciated IAC components. To investigate the robustness of the IAC network, the effects of pharmacological perturbation of the key IAC kinases FAK and Src on IACs were examined. FAK activity was inhibited with the small molecule inhibitor AZ13256675, and mass spectrometry-based protein quantification revealed that IAC protein composition was unaffected upon FAK inhibition. Moreover, IAC composition was also insensitive to Src inhibition using AZD0530 and to simultaneous FAK and Src inhibition. In contrast, phosphorylation of IAC components, cell migration and cell proliferation were reduced upon FAK and/or Src inhibition. These data suggest that IAC protein composition is robust to perturbation of key kinases, while flux of signals propagated through IACs via phosphorylation is kinase dependent. To examine IAC dynamics, the composition of IACs during IAC assembly and IAC disassembly were examined in the context of the meta-adhesome and consensus adhesome using IAC proteomic datasets. These analyses revealed the temporal dynamics of specific functional protein modules at IACs and detailed the compositional dynamics of the core cell adhesion machinery. In summary, these studies describe both a systems-level and a reductionist view of the IAC proteome, investigate the effects of kinase inhibition on IAC composition and chart IAC dynamics during their assembly and disassembly. These data demonstrate the usefulness of the meta-adhesome and consensus adhesome for future analyses of IAC proteomes.

L’incidence du mélanome a augmenté de façon considérable lors des trente dernières années avec un doublement tous les dix ans. Le mélanome ne représente que 5% des cancers cutanés mais entraîne 80% de décès, ce qui constitue un problème majeur de santé publique. En effet, cette tumeur est extrêmement agressive et possède un fort potentiel métastatique. Dès l’apparition de métastases, le pronostic vital devient fortement défavorable. Malgré des avancées thérapeutiques majeures, de nombreux patients sont encore réfractaires à ces nouveaux traitements. La compréhension des mécanismes impliqués dans le développement de cette tumeur reste donc un enjeu de premier ordre. Le séquençage d'exomes a conduit à l'identification d'une mutation dans le gène RAC1 (la mutation P29S) constituant une des mutations somatiques les plus fréquentes dans le mélanome (après les mutations BRAFV600, NRASQ61 et NF1). RAC1 est une petite GTPase qui fonctionne dans plusieurs processus cellulaires. Dans des conditions physiologiques, l'activité de RAC1 est principalement contrôlée par des protéines activatrices de l'activité GTPase (GAPs) et des facteurs d'échange Nucléotidique (GEF). GAPs et GEFs contrôlent le niveau de RAC1-GTP et régulent donc son activité. L'activité de RAC1 est aussi dépendante de son niveau d'expression protéique qui est contrôlé par des E3 ubiquitine ligases, parmi lesquelles HACE1. HACE1 est considérée comme un suppresseur de tumeur. De façon inattendue, les données obtenues montrent clairement que HACE1 favorise les propriétés migratoires et tumorigéniques des cellules de mélanome. / Melanoma incidence has considerably increased over the last thirty years, with a doubling every ten years. Melanoma accounts for only 5% of cutaneous cancers but causes more than 80% of deaths, which is a major public health problem. Indeed, this tumor is extremely aggressive and has a high metastatic potential. After the onset of metastases, the prognosis becomes highly unfavorable. Despite major therapeutic advances, many patients are still refractory to these new treatments. Understanding the mechanisms involved in the development of this tumor and the identification of new therapies remain a major issue. The sequencing of exomes led to the identification of a mutation in the RAC1 gene (P29S) constituting one of the most frequent somatic mutations in melanoma (after the BRAFV600, NRASQ61 and NF1 mutations). RAC1 is a small GTPase that is involved in several key cellular processes. Under physiological conditions, the activity of RAC1 is mainly controlled by GTPase activating proteins (GAPs) and Nucleotide Exchange (GEF) exchange factors. GAPs and GEFs control the level of RAC1- GTP and thus regulate its activity. The activity of RAC1 is also dependent on its protein level of expression which is controlled by E3 ubiquitin ligases, including HACE1. HACE1 is considered a tumor suppressor. Unexpectedly, our data clearly show that HACE1 promotes migratory and tumorigenic properties of melanoma cells. Indeed, inhibition of HACE1 alters migration of melanoma cells in vitro, as well as in vivo pulmonary colonization in mice. Transcriptomic analysis of 4 melanoma cell lines demonstrated that HACE1 suppression inhibits ITGAV and ITGB1 expression.