Dissertations / Theses on the topic 'Integrin alpha 5'

Le travail presente concerne l'etude du role joue par les deux motifs peptidiques npxy localises dans le domaine cytoplasmique de la sous-unite beta 1 des integrines. Par analogie avec le recepteur des ldl, ces deux motifs etaient assimiles au signal d'internalisation npxy permettant une association au complexe d'adaptines ha-2 et une internalisation dependante des puits a clathrine. En generant des lignees stables exprimant des integrines a sous-unite beta 1 mutees, nous avons montre que contrairement a d'autres recepteurs, les motifs npxy n'intervenaient pas dans l'internalisation de l'integrine alpha 5/beta 1. En revanche, nous avons observe que chacun des motifs npxy etait indispensable a l'assemblage des adherences focales. L'affinite pour le ligand extracellulaire n'etant pas affectee par les mutations realisees, nos resultats attribuent aux deux motifs npxy un role en tant que sites d'interaction, ou permettant la formation de sites d'interaction, avec une ou plusieurs proteines cytoplasmiques necessaires a l'assemblage des adherences focales. L'analyse de la fixation in vitro de la taline sur les chaines beta 1 mutees au niveau des sequences npxy, indique que cette proteine, pourtant necessaire a l'etablissement des adherences focales, n'est pas la proteine cible qui interagit avec les motifs npxy des integrines pour permettre leur recrutement au sein de ces adherences. La taline n'est pas suffisante a elle seule pour permettre l'assemblage des adherences focales. L'identite des proteines interagissant avec la sous-unite beta 1 de maniere dependante des motifs npxy, reste a determiner. L'immunoprecipitation des integrines a sous-unite beta 1 humaine dans des conditions menagees, a mis en evidence leur association avec diverses proteines cellulaires, controlee par le premier motif npxy (tyr 783). Ces proteines, non identifiees, pourraient jouer un role essentiel dans l'organisation des adherences focales, dependante du premier motif npxy de la sous-unite beta 1 des integrines, et/ou dans la signalisation cellulaire

Cellular adhesion plays a crucial role in the biological function of cells, allowing them to communicate and signal, as well as physically anchor, by enabling them to adhere to either other cells or the extra cellular matrix (ECM). This process is regulated by several factors including intrinsic bond kinetics, internal cellular signaling, environment, force exerted on the bond, and force history of the bond. Concerning the force and force history dependence, the observation of catch bonds in integrin binding has asked as more questions than it has answered. To explore the force and force history dependence this process, each bond was loaded to a peak force before relaxing to a much lower force that was held for the duration of the measurement. Two different integrins were studied, both of which have in previous works exhibited a catch bond. Furthermore, the effects of different metal ion conditions and an allosteric antagonist were also studied to elucidate the conformational effects on force priming of integrin. What was observed was that I domain, or αA domain, possessing integrin, whether tested against its more active or less active binding state, changed very little in terms of off rate once the priming force was applied. However in the I domain, or αA domain, lacking integrin, the observed off rate changed as well. It seems that force priming is capable of causing integrin to bind in a stronger manner regardless of the other conditions used to either activate or inhibit binding. However the way in which the binding is strengthened depends on the receptors structure.

Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding. The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length. These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin. The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions. The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer. On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.

Les integrines appartiennent a une grande famille de recepteurs heterodimeriques transmembranaires de type i impliques dans l'interaction des cellules entres elles ou a la matrice extracellulaire. Les signaux emis lors de l'engagement des integrines permettent le controle de nombreux processus cellulaires comme la migration, l'adherence, la proliferation, la differentiation ou encore l'apoptose. L'interaction des integrines avec leur ligand extracellulaire peut etre controlee depuis l'interieur de la cellule. Dans cette derniere regulation, le domaine cytoplasmique joue un role essentiel en interagissant avec des proteines intracellulaires. Dans la premiere partie de ce travail, j'ai pu montrer que la proteine kinase dependante du calcium et de la calmoduline de type ii (camkii) etait impliquee dans le controle de l'affinite de l'integrine 51. En effet, l'augmentation de l'activite de la camkii inhibe, in vitro, l'interaction entre l'integrine et la fibronectine. Ex vivo, l'inhibition de cette proteine kinase se traduit par une stimulation significative de l'etalement cellulaire en condition limitante de fibronectine. Par contre, l'expression ectopique de la camkii constitutivement active dans les cellules cho inhibe fortement leur etalement. Dans un second temps, j'ai etudie l'implication potentielle de la proteine icap-1 (integrin cytoplasmic domain associated protein-1) dans cette voie de regulation. Des mutations, au niveau du site potentiel de phosphorylation pour la camkii, abolissant ou au contraire mimant la phosphorylation perturbent severement l'etalement des cellules. Ceci montre que la proteine icap-1 et la camkii regulent l'etalement cellulaire dependant de l'integrine 51. Enfin, une approche moleculaire de l'action d'icap-1 a ete entreprise. Apres avoir confirme une interaction specifique entre la proteine icap-1 et l'integrine 51, j'ai analyse la localisation sub-cellulaire d'icap-1 a l'aide d'anticorps developpes au laboratoire. Aucune localisation de la proteine dans les adherences focales n'a pu etre mis en evidence. Par contre, la fixation d'icap-1 sur le domaine cytoplasmique des integrines a sous-unite 1 inhibe non seulement la fixation de la taline mais aussi le recrutement de proteines localisees dans les adherences focales. De ce fait, icap-1 pourrait etre un nouveau regulateur de la fonction adhesive des integrines.

A predisposição de recém-nascidos às doenças infecciosas é atribuída, em parte, a falta da memória imunológica pré-existente. Em recém-nascidos pré-termo, é presumido que o sistema imune seja menos desenvolvido ao nascimento, mas pouco se sabe sobre o tamanho e as características das subpopulações de linfócitos. Células T reguladoras (Treg) possuem papel crucial no controle do desenvolvimento de um sistema imune saudável incluindo a manutenção da autotolerância e, sua ausência, é responsável pela gama de manifestações inflamatórias e autoimunes observadas em pacientes com IPEX (Immunodeficiency, Poliendocrinopathy and Enteropathy X-linked Syndrome). Essas células são fenotipicamente caracterizadas pela presença do fator de transcrição Foxp3 (forkhead box P3) e pela alta expressão da cadeia ? do receptor de IL-2 (CD25), já que esta citocina é essencial para a geração, manutenção e funcionamento das células Treg. Pouco se sabe sobre a frequência destas células em recém-nascidos, particularmente em recém-nascidos muito prematuros ou moderados e recém-nascidos prematuros tardios, estudados como grupos separados. Resultados preliminares do nosso grupo revelaram uma maior capacidade dos recém-nascidos de produzir resposta pró-inflamatória em comparação aos adultos, a qual foi ainda mais acentuada pela diminuição da produção de IL-10, o que sugere uma função reguladora reduzida. Diante disso, o objetivo deste trabalho foi caracterizar fenotipicamente e quantificar a população de células Treg, por meio de citometria de fluxo, em sangue de cordão umbilical de 15 recém-nascidos pré-termo nascidos entre 30-336/7 semanas de gestação (Grupo 1), 19 recém-nascidos pré-termo nascidos entre 34-366/7 semanas de gestação (Grupo 2) e 20 recém-nascidos a termo nascidos entre 37-41 semanas de gestação (Grupo 3), todos clinicamente saudáveis e com peso adequado para a idade gestacional, em comparação com 26 adultos saudáveis. Os resultados demonstraram que existe uma correlação inversa entre a frequência de Treg e a idade gestacional, com frequências significativamente maiores de células Treg CD4+CD25hiCD127loFoxp3+ no Grupo 1 quando comparado aos Grupos 2 e 3 e no Grupo 2 comparado ao Grupo 3, assim como frequências e números de Treg mais elevados em todos os recém-nascidos comparados aos adultos. Todos os recém-nascidos exibiram maior frequência de células Treg com fenótipo naïve comparados aos adultos. A expressão de CTLA-4 nas células Treg naïve foi reduzida nos dois grupos de pré-termo comparados aos grupos de recém-nascidos a termo e adultos, assim como nas células Treg de memória do Grupo 1 comparado aos demais grupos. As frequências de Tregs alfa4beta7+ e alfa4beta1+ foram maiores em ambos os grupos de pré-termo, mas significativamente diferentes somente no Grupo 1, quando comparado aos recém-nascidos a termo e adultos. Em conclusão, foram observadas altas frequências de células Treg em recém-nascidos pré-termo e a termo, e essas frequências mostraram correlação inversa com a idade gestacional. Essas células exibiram um perfil naïve quando comparadas às dos adultos, com alta expressão de CD45RA e alfa4beta7+ e menor expressão de CTLA-4, sugerindo uma menor função, particularmente em recém-nascidos muito prematuros
The predisposition of newborn infants to infectious diseases is attributed, in part, to the lack of pre-existing immunological memory. In preterm newborns, it is assumed that the immune system is less developed at birth, but little is known about the size and characteristics of lymphocyte subpopulations. Regulatory T cells (Tregs) have a crucial role in controlling the development of a healthy immune system including the maintenance of self-tolerance and, their absence, is responsible for the range of inflammatory and autoimmune manifestations observed in patients with IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked Syndrome). These cells are phenotypically characterized by the presence of the transcription factor Foxp3 (forkhead box P3) and by the high expression of the ? chain of the IL-2 receptor (CD25), as this cytokine is essential for the generation, maintenance and function of Treg cells. Little is known about the frequency of these cells in neonates, particularly in very and moderate preterm newborns and late preterm newborns studied as separate groups. Preliminary results from our group revealed greater ability of newborns to produce proinflammatory response compared to adults, which was further accentuated by the decreased production of IL-10, which suggests a reduced regulatory function. Thus, the aim of this study was to phenotypically characterize and quantify the population of Treg cells, by flow cytometry, in the cord blood of 15 preterm newborns born at 30-336/7 gestation weeks (Group 1), 19 preterm newborns born at 34-366/7 gestation weeks (Group 2) and 20 term newborns born at 37-41 gestation weeks (Group 3), all clinically healthy and adequate-for-gestational-age, compared to 26 healthy adults. The results demonstrated that there is an inverse correlation of the Treg frequency and gestational age, with significantly higher frequencies of CD4+CD25hiCD127loFoxp3+ Treg cells in Group 1 compared to Groups 2 and 3 and in Group 2 compared to Group 3, as well as significantly higher Treg frequencies and numbers in all the neonates compared to the adults. All of the newborns exhibited increased Treg frequencies with a naive phenotype compared to the adults. CTLA-4 expression in the naive Tregs was decreased in both preterm groups compared with those from term newborns and adults, as well as in the memory Treg cells from Group 1 compared with the other groups. The frequencies of alfa4beta7+ and alfa4beta1+ Tregs were higher in both preterm groups, but significantly different only in Group 1, when compared with those from the term newborns and the adults. In conclusion, high frequencies of Tregs were observed in term and preterm newborns, and these frequencies showed an inverse correlation with gestational age. These cells exhibited a naive profile when compared with adults, with high expression of CD45RA and alfa4beta7+ and lower expression of CTLA-4, implying a decreased function, particularly in very preterm newborns

Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding. The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length. These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin. The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions. The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer. On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.

碩士
國立成功大學
生物化學研究所
92
Integrins are a family of cell surface receptors that mediate cell cellular adhesion, cell migration and signal transduction. It is known that integrins play important roles in the initiation and progression of many common diseases. Therefore, antagonism of integrins provides an approach for the treatment and prevention of these diseases. The RGD sequence is the cell attachment site of a large number of adhesive ECM, blood, and cell surface proteins and nearly half of 24 known integrins recognize this sequence in their adhesive ligands. Many studies have showed that the amino acid residues flanking the RGD motif of snake venom disintegrins control their binding specificity. Rhodostomin (Rho) is a potent integrin inhibitor and contains a PRGDMP sequence at positions of 48-53. Based on our results of cell adhesion analyses, we found that Rho mutants containing either ARGDGP or PRGDGP sequence can selectively inhibit integrin alpha v beta 3 and Rho mutants containing ARGDXP sequence in the RGD loop have better inhibitory activity to integrin alpha 5 beta 1. To understand structural determinants of integrins alpha v beta 3 and alpha 5 beta 1 recognition, we have determined 3D structures of these mutant proteins (ARGDMP, ARGDNP, ARGDGP and PRGDGP) by using NMR spectroscopy. Based on our structural analyses, the tertiary folds of wild-type and mutant proteins are the same. However, the backbone conformation of the RGD loop and the side-chain orientation of the D51 residue of mutant proteins (ARGDGP and PRGDGP) differ from those of wild-type Rho. Their structures are similar to our reported structure of the ARGDDL mutant, an integrin alpha v beta 3-specific protein. These results suggest that these conformational differences may be responsible for their inhibitory selectivity to integrin alpha v beta 3. In addition, model-free analyses of PRGDMP and ARGDMP proteins reveal that mutation of P48 to A caused a decrease in the order parameter (S2) of the R49 residue of the ARGDMP protein, mainly due to the decrease of R2. This difference may be responsible for the better inhibitory activity of Rho mutants containing ARGDXP sequence to integrin alpha 5 beta 1. Using 3D structure of alpha v beta 3 integrin as template, 3D model structures of integrins alpha IIb beta 3 and alpha 5 beta 1 were generated by using homology modeling. Docking integrins alpha IIb beta 3, alpha v beta 3, and alpha 5 beta 1-specific Rho mutants to their receptors will be used to identify their possible interacting amino acids. Our findings suggest that the residues flanking the RGD motif of disintegrins regulate their structure, dynamics and function. These results will be used to design potent integrins alpha IIb beta 3, alpha v beta 3, and alpha 5 beta 1-specific antagonists.