Дисертації з теми "Integrin affinity"

Recent work has shown that the transmembrane protein CD98 is able to influence the affinity with which β1 integrins bind to extracellular ligands. The first part of this thesis presents confocal microscopy and co-immunoprecipitation experiments that confirm the physical juxtaposition of the two proteins within the cell membrane, suggesting a direct functional link between the two. It also demonstrated that cross-linking CD98 stimulates both phosphoinositide 3-kinase intracellular signalling and increased β1 integrin-dependent cellular adhesion. Because of the role of CD98 in integrin affinity modulation, the immunohistochemical expression of CD98 and its ligand, galectin-3, was studied in a variety of human ling diseases including lung cancers. The major finding of this work was a striking distinction between high expression of galectin-3 in non-small cell lung cancer and low expression in small cell lung cancer. This may hag significant implications for the differing clinical behaviours of these two groups of cancers. The final section of this thesis returns to describe experiments aimed at defining the molecular regulators of integrin affinity more clearly. A genetic screen of a cDNA library was undertaken to identify candidate genes coding for proteins able to rescue integrins from the low affinity state induced by the small signalling protein H-Ras. This identified a candidate cDNA 480, recognised to be part of a novel gene Nessie, which codes for a large protein with multiple transmembrane domains. Both 480 and Nessie appear to have the ability to rescue integrin affinity from H-Ras suppression.

Integrins are heterodimeric transmembrane proteins that provide a bi-directional link between the cell’s internal biological mechanisms and the extracellular environment. During inside-out signalling, intracellular messages converge on the integrin cytoplasmic domain to induce a conformational change. This is transmitted to the extracellular domain where it results in an alteration in affinity for integrin ligands such as fibronectin and laminin. In this way the cell has developed the ability to modulate the critical functions of adhesion and cell movement. In outside-in signalling, the integrin performs a more complex function than simple adhesion; upon binding to ligand, the integrin extracellular domain undergoes a conformational change which is transmitted to the cytoplasmic domain. This alters the integrin’s cytoplasmic domain affinity for intracellular signalling proteins and results in the activation of intracellular second messenger pathways. In this way, the extracellular milieu is able to influence intracellular signalling including those involved in apoptosis. This thesis demonstrates data which provide original insights into bi-directional integrin signalling: Inside-out signalling: Constitutively active Notch1 increases β3-integrin affinity and abrogates Hras-mediated integrin suppression without increasing expression of β3- integrin. Dominant-Negative Rras blocks Notch-mediated integrin activation and Notch1-mediated reversal of Hras and Raf-mediated integrin suppression and this is independent of erk phosphorylation. Notch1 induces Rras activation. Functional adhesion assays confirm that Notch1IC increases K562 adhesion in a β1-integrin dependent manner and this is abrogated by Dominant-Negative Rras. This data supports a mechanism in which Notch1 increases integrin affinity via activation of Rras. Outside-in signalling: Evidence is presented demonstrating that extracellular matrix proteins, laminin and fibronectin, activate β1-integrins to protect SCLC cells against the apoptotic effects of etoposide and ionizing radiation via PI3Kinase activation. This occurs in two ways: 1) PI3Kinase-dependent β1-integrin signalling resulting in phosphorylation of Bad and reduced caspase-9 cleavage and 2) a β1-integrinmediated over-riding of etoposide and radiotherapy-induced cell cycle S phase delay and G2/M arrest. β1-integrin-mediated outside-in survival signalling was investigated further in the in vivo setting; MatrigelTM, a basement membrane product rich in extracellular matrix proteins, promoted SCLC xenograft survival and growth in a β1-integrin and tyrosine kinase-dependent manner. This data provides novel insights into the critical functions that integrins play in adhesion and survival signalling.

In this thesis I have sought to understand the mechanism by which H-Ras and its effectors modulate integrin affinity. H-Ras is a member of the Ras superfamily of small GTP binding proteins. Expression of the constitutively active variant of H-Ras (Ras G12V) within an integrin affinity reporter system (αβ-py cells) reduced integrin affinity (suppressed integrin). Ras effector mutants revealed that integrin suppression is mediated by Raf-dependent and Raf-independent signalling pathways. Raf-independent signalling pathways activated by Ral-GEFs and PI3-kinase were not recognisable for integrin suppression. An active variant of R-Ras (R-Ras G38V) reversed integrin suppression by both Raf-dependent and - independent pathways, indicating that these pathways may converge at a point proximal to the integrin. Raf initiates a protein signalling cascade leading to ERK activation that is responsible for many of the Ras/Raf-dependent biological functions. However, Raf-dependent integrin suppression was insensitive to MEK inhibition with the PD098059 compound. A novel Raf mutant (T481A) that fails to bind to MEK was also capable of mediating integrin suppression in the absence of ERK activation. Surprisingly, Raf-BxB T481A-mediated integrin suppression was sensitive to expression of MKP-1. Taken together it is proposed that Raf may mediate integrin suppression via a MEK-independent pathway that may utilise a member of the MAP kinase superfamily. In conclusion, integrin suppression by Ras is mediated by both Raf-independent and dependent pathways. Signalling by Raf may utilise components other than those present in the classical Ras to ERK protein cascade.

The impact of monoclonal antibodies (mAbs) in current pharmaceutical research is due to their unique ability to bind biological targets with very high affinity. On the other hand, there is a considerable interest in the development of small molecule ligands with antibody-like affinities, which may overcome some limitations of mAbs. This PhD work describes the development of general strategies to increase the binding affinity of peptide ligands bearing the Arg-Gly-Asp motif, i.e. the well-known recognition sequence of specific tumor-associated integrin receptors. In our first approach, we designed a bicyclic peptide bearing two RGD motifs that displayed an enhanced inhibition of ECM protein binding to integrin receptors αvβ3 and α5β1 and marked biological effects in U-373 MG glioblastoma cells. Later on, we focused on the 2-hydroxybenzaldehyde tag (2HB), which can engage ϵ‐amino groups of Lys residues in stable imines. After investigating the 2HB installation to different types of reactive handles, we conjugated the 2HB tag to the N-side and on the C-side of a cyclic RGD peptide. The resulting conjugates have been investigated as novel αvβ3 integrin ligands and the nature of the ligand-protein interaction has been investigated performing in silico experiments. For both the 2HB-RGD conjugates, the biological results and the computational outcomes demonstrated to be coherent with each other, proving the feasibility of the reversible covalent engagement of Lys residues with the 2HB tag to enhance the affinity of a well-known small ligand.

A l'aide de cellules cho15b bloquees en prometaphase par du nocodazole, nous avons etudie la fonction du recepteur de la fibronectine exprime a la surface de cellules mitotiques. Une etude par cytometrie en flux a l'aide d'un anticorps dirige contre cette integrine, et des etudes de fixation de fibronectine radiomarquee sur la membrane cellulaire, montrent qu'un nombre constant d'integrines est exprime a la surface cellulaire au cours du cycle cellulaire. De plus, le recepteur de la fibronectine est toujours dans un etat de haute affinite pour son ligand soluble ou insoluble au cours de la mitose. Ces resultats indiquent que l'arrondissement des cellules observe durant la mitose ne resulte pas d'une parte de l'affinite du recepteur pour son ligand extracellulaire ; ce changement morphologique serait plutot la consequence d'une desorganisation des plaques d'adherence. A partir d'un lysat de cellules cho15b, nous avons mis au point un test in vitro afin d'etudier l'interaction de la fibronectine avec le recepteur de la fibronectine dans un environnement cytosolique. Dans ce test, l'interaction integrine/fibronectine necessite du calcium intracellulaire, la calmoduline, et une activite phosphatase. De plus, l'action d'inhibiteurs specifiques de phosphatases et l'inhibition de l'interaction integrine/fibronectine par un anticorps dirige contre la calcineurine, la phosphatase dependante du calcium et de la calmoduline, suggerent que la calcineurine permet l'interaction entre le recepteur de la fibronectine et son ligand. Des experiences de marquage metabolique montrent que l'integrine elle-meme n'est pas la cible d'une cascade de phosphorylation/dephosphorylation impliquant la calcineurine et modulant l'affinite de l'integrine. Ces resultats montrent qu'in vitro, un substrat de la calcineurine regule l'affinite du recepteur de la fibronectine en interagissant avec un effecteur non-identifie

Social network sites such as MySpace and Facebook play an important role in mediating the everyday social and cultural lives of many internet users. Young internet users were amongst the first to incorporate these sites into their everyday lives, and many young people continue to use them to connect and share with their networks, forging conventions and strategies for ‘being’ in online social spaces. For some of these young people, participation in these social spaces has become central for inclusion amongst peer groups. These sites offer a platform of mediated sociality that is distinct, while also manifesting in forms of interaction that are familiar and embedded in the everyday, blurring distinctions between online and offline, and troubling notions of public and private. Drawing on qualitative data collected between mid-2009 and late-2010, this thesis charts the role of the two most dominant social network sites, MySpace and Facebook, in the social lives of thirty-three young people in Australia. Fieldwork was conducted in two phases: first, through gaining access to the profiles of my participants, observing interactions and exchanges on these profiles, and analysing content; and second, drawing on these observations to frame semi-structured, in-depth, in-person interviews. At the centre of the analysis of my findings is a focus on questions of identity and self-presentation online, and how the performance of identity in online social spaces represents a reflexive ordering of self-narratives that manifest in a ‘digital trace’. I explore friending strategies, notions of integrity and authenticity, and challenge dominant conceptualisations of belonging that do not adequately encompass the systems of belonging made visible by my participants on social network sites.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Humanities
Arts, Education and Law
Full Text

A afinidade eletrônica (AE) é uma importante propriedade de átomos e moléculas, sendo definida como a diferença de energia entre a espécie neutra e seu respectivo íon negativo. Uma vez que a AE é uma fração muito pequena da energia eletrônica total das espécies neutra e aniônica, é necessário que tais energias sejam determinadas com elevado grau de precisão. A receita utilizada para o cálculo teórico acurado da AE atômica e molecular baseia-se na escolha de um conjunto adequado de funções de base juntamente com o emprego de teorias com altos níveis de correlação eletrônica. Durante o cálculo, o mesmo conjunto de base é utilizado para descrever o elemento neutro e seu respectivo ânion. Geralmente, os conjuntos de base para descrever propriedades de ânions possuem seus expoentes otimizados em ambiente neutro, e sua difusibilidade é conferida pela adição de funções difusas para cada valor de momento angular, l. A ideia deste trabalho está no desenvolvimento de conjuntos de base otimizados exclusivamente em ambiente aniônico para cálculos precisos de afinidade eletrônica. Deste modo, foram escolhidos os átomos para serem estudados: B, C, O e F. Os conjuntos de base foram gerados pelo Método da Coordenada Geradora Hartree-Fock, empregando a técnica da Discretização Integral Polinomial para a solução das integrais do problema. Os conjuntos de base obtidos são compostos por (18s13p) primitivas que foram contraídos para [7s6p] via esquema de contração geral proposto por Raffenetti. Os conjuntos contraídos foram polarizados para 4d3f2g e 4d3f2g1h, sendo os expoentes otimizados em ambiente CISD através do método SIMPLEX. Avaliaram-se as funções de base no cálculo de afinidades eletrônicas, tendo seus resultados comparados aos obtidos utilizando as bases aug-cc-pVQZ e aug-cc-pV5Z. A análise dos resultados demonstrou que os conjuntos de base difusos, gerados neste trabalho, reproduzem de maneira satisfatória as afinidades eletrônicas em relação ao valor experimental. Os conjuntos difusos polarizados para 4d3f2g1h apresentaram eficiência superior aos conjuntos aug-cc-pVQZ e, em alguns casos, aos conjuntos aug-cc-pV5Z que são consideravelmente maiores.
The electron affinity (EA) is an important property of atoms and molecules defined as the energy difference between the neutral species and its negative ion. Since the EA is a very small fraction of the total electronic energy of anionic and neutral species, one must determine these energies with high accuracy. The recipe used to calculate accurate atomic and molecular EAs is based on the choice of an adequate basis set and the use of high level of electron correlation calculations. In the computation of EAs, the same basis set is used to describe both neutral and negatively charged species. In general, the basis sets designed to describe anionic properties have their exponents optimized in neutral environment, and its diffuseness is acquired through the addition of diffuse functions for each angular momentum. The main idea of this work is to develop basis sets optimized exclusively in anionic environment that would be applied in accurate calculations of electron affinity. Thus, here follows the chosen atoms to be studied: B, C, O and F. The basis sets were generated by the Generator Coordinate Hartree-Fock Method through the Polynomial Integral Discretization Method. Basis sets were obtained containing (18s13p) primitives that were contracted to [7s6p] via Raffenetti\'s general contraction scheme. The contracted basis sets were polarized to 4d3f2g and 4d3f2g1h, and the exponents of polarization were optimized in a CISD environment through the Simplex algorithm. The basis sets quality was evaluated through the calculation of the electron affinities. The results were compared to those obtained by using the aug-cc-pVQZ and aug-cc-pV5Z basis-sets. The calculation showed that our diffuse basis sets reproduce satisfactorily the electron affinities when compared to the experimental data. The diffuse basis sets polarized to 4d3f2g1h showed to be more efficient than the aug-cc-pVQZ basis sets and in some cases also better than the aug-cc-pV5Z basis sets that are considerably larger.

Alternative scaffolds are non-antibody proteins that can be engineered to bind new targets. They have found useful niches in the therapeutic space due to their smaller size and the ease with which they can be engineered to be bispecific. We sought a new scaffold that could be used for therapeutic ends and chose the C2 discoidin domain of factor VIII, which is well studied and of human origin. Using yeast surface display, we engineered the C2 domain to bind to αvβ3 integrin with a 16 nM affinity while retaining its thermal stability and monomeric nature. We obtained a crystal structure of the engineered domain at 2.1 Å resolution. We have christened this discoidin domain alternative scaffold the “discobody.”

碩士
國立臺灣大學
生化科技學系
105
According to WHO cancer statistics in 2012, prostate cancer is the second most common cancer and the fifth leading cause of cancer death in men around the world. Eighty percent of men with the localized prostate cancer are still alive after diagnosis in 5 years, however, at the distant stage of prostate cancer, cancer that have spread to other organs, the survival rate drop dramatically and is the most causes of prostate cancer death. Therefore, it is an urgent need to develop precise and non-invasion diagnostic method for metastatic prostate cancer. The integrin α2β1 is the major type I collagen-binding receptor, and it is overexpressed in many cancer types. The importance of integrin α2β1 involved in prostate cancer metastasis has made it an appropriate biomarker for prostate cancer detection. Hence, in this thesis, the goal was to identify integrin α2β1-binding peptides and ultimately develop as the imaging agent for metastatic prostate cancer diagnosis. First, the recombinant integrin α2 I domain protein was purified and used for selection of high affinity binding peptides with the loop-constrained heptapeptides (C7C) phage library by biopanning. After measurement of the relative binding affinity by phage ELISA binding assay, two integrin α2 I domain-binding phages (C7, C8) were identified. The result of type I collagen competition assay indicated that the C7 phage, but not the C8 phage, could compete with type I collagen to bind to integrin α2 I domain. Moreover, the dissociation constant (Kd) of C7 or C8 peptide to integrin α2 I domain was determined by FITC-peptide saturation binding assay, and the Kd of C7 and C8 peptides were 7 μM and 12 μM, respectively. Furthermore, we used two peptides to target the two prostate cancer cell lines (PC-3 and 22Rv1) that have different integrin α2β1 expression level. By using FITC-labeled C7 peptide or FITC-labeled C8 peptide incubating with these two cell lines and followed by IN Cell Analyzer 2000, we demonstrated that two peptides had greater tendency of binding to PC-3 cells than 22Rv1 cells. In conclusion, C7 or C8 peptide may have potential to be developed as molecular imaging tracer in prostate cancer diagnosis.

碩士
國立臺灣大學
生化科技學系
106
Molecular imaging is a powerful method and technique in precision medicine. With a labeled probe which is specific to the corresponding biomarker, people could easily detect, locate and diagnose disease in a more accurate way. Integrin α2β1 was identified as a major collagen binding receptor. Previous research indicated that prostate and colon cancers which overexpressed integrin α2β1 are more possible to metastasize to bone marrow and liver because of abundant collagen in the microenvironment. Therefore, the purpose of this research is to identify the DNA aptamer which could specifically bind to Integrin α2β1 and used as a probe in molecular imaging. SELEX (Systematic evolution of ligands by exponential enrichment) is the technique for producing high affinity aptamer. The aptamer library would incubate with target molecules for selection. After several rounds of selection, the remaining aptamer could specifically bind to target molecule. In this thesis, two strategies for single-stranded DNA generation were used in SELEX: asymmetric PCR and streptavidin beads with alkaline treatment. However, the strategy of asymmetric PCR was time-consuming and hard to control as a result we did not finish our selection with this method. The binding affinity of two aptamer candidates, YCP-11 and YCP-12, were tested by saturation binding assay but the result showed that these aptamers were not specific binding to our target protein. Further, from the sequencing result of different round aptamer pool, we found that the losing of library diversity might be the main reason that led to the failure of SELEX. We thought that two possible reasons might cause the losing of library diversity: the concentration of aptamer was low in the selection step or the washing condition was too harsh. We suggested that increasing the concentration of aptamer to 500 nM and adjusting washing condition to 200 μl in volume with 30 seconds every time might improve the present method of SELEX.

I sistemi di trasduzione del segnale sono finemente regolati dall’equilibrio tra le attività di fosforilazione e defosforilazione. Come recentemente dimostrato, le tirosina chinasi della famiglia JAK sono in grado di modulare l’affinità integrinica indotta da chemochine e il conseguente homing negli organi linfoidi secondari di linfociti T umani. Tuttavia, il ruolo delle tirosina fosfatasi nel contesto del reclutamento leucocitario è ancora per lo più inesplorato. Scopo di questa tesi è stato quello di chiarire il ruolo della defosforilazione nel contesto dell’attivazione integrinica, focalizzando l’attenzione sulla proteina fosfatasi in tirosina recettoriale di tipo gamma (PTPRG), una fosfatasi altamente espressa nei monociti umani primari. Mediante lo sviluppo di un nuovo approccio metodologico, abbiamo scoperto che l’attivazione di PTPRG blocca l’induzione dell’alto stato di affinità dell’integrina LFA-1. Analisi fosfoproteomica e computazionale hanno dimostrato che l’attivazione di PTPRG interferisce sullo stato di fosforilazione di almeno 31 proteine coinvolte nella trasduzione del segnale. Uno studio più approfondito ha inoltre confermato che le JAK chinasi sono direttamente coinvolte nell’adesione monocitaria mediata dalle integrine, e che l’attivazione di PTPRG svolge un ruolo anti-adesivo, inducendo la defosforilazione delle tirosine 1007 e 1008 di JAK2, critiche per la sua autofosforilazione attivatoria. Nel complesso, i dati dimostrano non solo come questo nuovo approccio tecnologico sia adatto allo studio del ruolo delle fosfatasi recettoriali, ma anche che, defosforilando JAK2, PTPRG inibisce la rapida attivazione dell’affinità integrinica nei monociti umani primari. Questo studio propone quindi un nuovo potenziale ruolo di PTPRG come regolatore del traffico leucocitario.
Regulation of signal transduction networks depends on protein kinase and phosphatase activities. Protein tyrosine kinases of the JAK family have been shown to regulate integrin affinity modulation by chemokines and mediated homing to secondary lymphoid organs of human T-lymphocytes. However, the role of protein tyrosine phosphatases in leukocyte recruitment is still elusive. Here we address this issue by focusing on protein tyrosine phosphatase, receptor type γ (PTPRG), a tyrosine phosphatase highly expressed in human primary monocytes. We developed a novel methodology to study the signaling role of receptor type tyrosine phosphatases and found that activated PTPRG blocks chemoattractant-induced β2 integrin activation. Specifically, triggering of LFA-1 to high affinity state is prevented by PTPRG activation. High-throughput phospho-proteomics and computational analyses show that PTPRG activation affects the phosphorylation state of at least 31 signaling proteins. Deeper examination shows that JAKs are critically involved in integrin-mediated monocyte adhesion and that PTPRG activation leads to JAK2 de-phosphorylation on the critical 1007-1008 phosphotyrosine residues, implying JAK2 inhibition and, thus, explaining the anti-adhesive role of PTPRG. Overall, the data validate a new approach to study receptor tyrosine phosphatases and show that, by targeting JAKs, PTPRG down-modulates the rapid activation of integrin affinity in human monocytes, thus emerging as a potential novel critical regulator of leukocyte trafficking.