Biological Signatures of Vaccine Responses

The set of host- and pathogen-specific molecular features of a disease comprise its “signature”. We hypothesize that biological signatures enables distinctions between vaccinated vs. infected individuals. In our research, using porcine samples, protocols were developed that could also be used to identify biological signatures of human disease. Different classes of molecular features will be tested during this project, including indicators of basic immune capacity, which are being studied at this instance. These indicators of basic immune response such as porcine cytokines and antibodies were validated using Enzyme-linked immunosorbent assay (ELISA). This is an established method that detects antigens by their interaction with a specific antibody coupled to a polystyrene substrate. Serum from naïve and vaccinated pigs was tested for the presence of cytokines. We were able to differentiate the presence of porcine IL-6 in normal porcine serum with or without added porcine IL-6 by ELISA. In addition, four different cytokines were spotted on a grating-coupled surface plasmon resonance imaging system (GCSPRI) chip and antibody specific for IL-8 was run over the chip. Only the presence of IL-8 was detected; therefore, there was no cross-reactivity in this combination of antigens and antibodies. This system uses a multiplexed sensor chip to identify components of a sample run over it. The detection is accomplished by the change in refractive index caused by the interaction between the antibody spotted on the sensor chip and the antigen present in the sample. As the multiplexed GCSPRI is developed, we will need to optimize both sensitivity and specificity, minimizing the potential for cross-reactivity between individual analytes. The next step in this project is to increase the sensitivity of detection of the analytes. Currently, we are using two different antibodies (that recognize a different part of the antigen) to amplify the signal emitted by the interaction of antibody with its cognate antigen. The development of this sensor chip would not only allow to detect FMD virus, but also to differentiate between infected and vaccinated individuals, on location. Furthermore, the diagnosis of other diseases could be done with increased accuracy, and in less time due to the microarray approach.

Research lines in Hyperthermia at the Bioinstrumentation Laboratory of the Centre for Biomedical Technology

The Bioinstrumentation Laboratory belongs to the Centre for Biomedical Technology (CTB) of the Technical University of Madrid and its main objective is to provide the scientific community with devices and techniques for the characterization of micro and nanostructures and consequently finding their best biomedical applications. Hyperthermia (greek word for “overheating”) is defined as the phenomenon that occurs when a body is exposed to an energy generating source that can produce a rise in temperature (42-45ºC) for a given time [1]. Specifically, the aim of the hyperthermia methods used in The Bioinstrumentation Laboratory is the development of thermal therapies, some of these using different kinds of nanoparticles, to kill cancer cells and reduce the damage on healthy tissues. The optical hyperthermia is based on noble metal nanoparticles and laser irradiation. This kind of nanoparticles has an immense potential associated to the development of therapies for cancer on account of their Surface Plasmon Resonance (SPR) enhanced light scattering and absorption. In a short period of time, the absorbed light is converted into localized heat, so we can take advantage of these characteristics to heat up tumor cells in order to obtain the cellular death [2]. In this case, the laboratory has an optical hyperthermia device based on a continuous wave laser used to kill glioblastoma cell lines (1321N1) in the presence of gold nanorods (Figure 1a). The wavelength of the laser light is 808 nm because the penetration of the light in the tissue is deeper in the Near Infrared Region. The first optical hyperthermia results show that the laser irradiation produces cellular death in the experimental samples of glioblastoma cell lines using gold nanorods but is not able to decrease the cellular viability of cancer cells in samples without the suitable nanorods (Figure 1b) [3]. The generation of magnetic hyperthermia is performed through changes of the magnetic induction in magnetic nanoparticles (MNPs) that are embedded in viscous medium. The Figure 2 shows a schematic design of the AC induction hyperthermia device in magnetic fluids. The equipment has been manufactured at The Bioinstrumentation Laboratory. The first block implies two steps: the signal selection with frequency manipulation option from 9 KHz to 2MHz, and a linear output up to 1500W. The second block is where magnetic field is generated ( 5mm, 10 turns). Finally, the third block is a software control where the user can establish initial parameters, and also shows the temperature response of MNPs due to the magnetic field applied [4-8]. The Bioinstrumentation Laboratory in collaboration with the Mexican company MRI-DT have recently implemented a new research line on Nuclear Magnetic Resonance Hyperthermia, which is sustained on the patent US 7,423,429B2 owned by this company. This investigation is based on the use of clinical MRI equipment not only for diagnosis but for therapy [9]. This idea consists of two main facts: Magnetic Resonance Imaging can cause focal heating [10], and the differentiation in resonant frequency between healthy and cancer cells [11]. To produce only heating in cancer cells when the whole body is irradiated, it is necessary to determine the specific resonant frequency of the target, using the information contained in the spectra of the area of interest. Then, special RF pulse sequence is applied to produce fast excitation and relaxation mechanism that generates temperature increase of the tumor, causing cellular death or metabolism malfunction that stops cellular division

Epitaxial self-alignment: A new route to hybrid active plasmonic nanostructures

Concepts of lateral ordering of epitaxial semiconductor quantum dots (QDs) are for the first time transferred to hybrid nanostructures for active plasmonics. We review our recent research on the self-alignment of epitaxial nanocrystals of In and Ag on ordered one-dimensional In(Ga)As QD arrays and isolated QDs by molecular beam epitaxy. By changing the growth conditions the size and density of the metal nanocrystals are easily controlled and the surface plasmon resonance wavelength is tuned over a wide range in order to match the emission wavelength of the QDs. Photoluminescence measurements reveal large enhancement of the emitted light intensity due to plasmon enhanced emission and absorption down to the single QD level.

Contribución al estudio de sistemas de hipertermia óptica y su aplicación en muestras biológicas

Las nanopartículas de metales nobles (especialmente las de oro) tienen un gran potencial asociado al desarrollo de sistemas de terapia contra el cáncer debido principalmente a sus propiedades ópticas, ya que cuando son irradiadas con un haz de luz sintonizado en longitud de onda con su máximo de Resonancia de Plasmón Superficial, absorben de manera muy eficiente dicha luz y la disipan rápidamente al medio en forma de calor localizado. Esta característica por tanto, puede ser aprovechada para conseguir elevar la temperatura de células tumorales hasta sobrepasar umbrales a partir de los cuales se produciría la muerte celular. Partiendo de estos principios, esta tesis se centra en el desarrollo y la caracterización de una serie de prototipos de hipertermia óptica basados en la irradiación de nanopartículas de oro con un haz de luz adecuado, así como en la aplicación in vitro de la terapia sobre células cancerígenas. Además, el trabajo se orienta a identificar y comprender los procesos mecánicos y térmicos asociados a este tipo de hipertermia, y a desarrollar modelos que los describan, estudiando y planteando nuevas formas de irradiación, para, en última instancia, poder optimizar los procesos descritos y hacerlos más efectivos. Los resultados obtenidos indican que, el uso de nanopartículas de oro, y más concretamente de nanorods de oro, para llevar a cabo terapias de hipertermia óptica, permite desarrollar terapias muy efectivas para inducir muerte en células cancerígenas, especialmente en tumores superficiales, o como complemento quirúrgico en tumores internos. Sin embargo, los efectos de la toxicidad de las nanopartículas de oro, aún deben ser detalladamente estudiados, ya que este tipo de terapias sólo será viable si se consigue una completa biocompatibilidad. Por otro lado, el estudio exhaustivo de los procesos térmicos que tienen lugar durante la irradiación de las nanopartículas ha dado lugar a una serie de modelos que permiten determinar la efectividad fototérmica de las nanopartículas y además, visualizar la evolución de la temperatura tanto a escala nanométrica como a escala macrométrica, en función de los parámetros ópticos y térmicos del sistema. El planteamiento de nuevas formas de irradiación y el desarrollo de dispositivos orientados a estudiar los fenómenos mecánicos que tienen lugar durante la irradiación pulsada de baja frecuencia y baja potencia de nanopartículas de oro, ha dado lugar a la detección de ondas de presión asociadas a procesos de expansión termoelástica, abriendo la puerta al desarrollo de terapias de hipertermia que combinen la muerte celular producida por calentamiento con la muerte derivada de los fenómenos mecánicos descritos.VII Noble metal nanoparticles (especially gold ones), have a huge potential in the development of therapy systems against cancer mainly due to their optical properties, so that, when these particles are irradiated with a light that is syntonized in wavelength with their maximum of Surface Plasmon Resonance, they effectively absorb and dissipate the light to the surrounding medium as localized heat. We can take advantage of this characteristic for rising the temperature of cancer cells above the threshold at which cellular death would occur. From these principles, this thesis is oriented to the development and characterization of a series of optical hyperthermia prototypes based on the irradiation of gold nanoparticles using the suitable light, and on the in vitro application of this therapy over cancer cells, to understand the mechanical and thermal processes associated with this kind of hyperthermia, developing descriptive models, and to study and to approach new ways of irradiation in order to, ultimately, optimize the described processes and make them more effective. The obtained results show that, the use of gold nanoparticles, and more specifically, of gold nanorods, to carry out optical hyperthermia therapies, allows the development of very effective therapies in order to induce death in VIII cancer cells, especially in superficial tumors, or like surgical complement in more internal tumors. However, the toxicity effects of the gold nanoparticles still need to be studied more detail, because this kind of therapies will be feasible only if a complete biocompatibility is achieved. On the other hand, the exhaustive study of the thermal processes that take place during the irradiation of the nanoparticles resulted in a series of models that allow the determination of the photothermal efficiency of the nanoparticles and also the visualization of the temperature evolution, both at nanoscale and at macroscale, as a function of the optical and thermal parameters of the system. The proposal of new ways of irradiation and the development of devices oriented to study the mechanical effects that take place during the low frequency and low power pulsing irradiation of gold nanoparticles has led to the detection of pressure waves associated to thermoelastic expansion processes, opening the door to the development of hyperthermia therapies that combine the cellular death due to the heating with the death derived from the described mechanical phenomena.

Selectively filled photonic liquid crystal fibers = Fibras de cristal fotónico selectivamente llenas con cristal líquido

El presente trabajo de Tesis se ha centrado en el diseño, fabricación y caracterización de dispositivos basados en fibras de cristal fotónico infiltrado selectivamente con cristales líquidos, polímeros y una mezcla de ambos. Todos los dispositivos son sintonizables, y su área de aplicación se centra en comunicaciones ópticas y sensores. La manipulación y fusionado de fibras fotónicas, el llenado selectivo de determinadas cavidades y la alineación recíproca de fibras mantenedoras de polarización son tareas muy específicas y delicadas para las que se requieren protocolos muy estrictos. Previo a la fabricación de dispositivos ha sido necesaria por tanto una tarea de sistematización y creación de protocolos de fabricación. Una vez establecidos se ha procedido a la fabricación y caracterización de dispositivos. Los dispositivos fabricados se enumeran a continuación para posteriormente detallar una a una las singularidades de cada uno. • Interferómetros intermodales hechos a partir de una porción de fibra fotónica soldada entre dos fibras estándar, bien monomodo o PANDA (mantenedora de polarización). Estos interferómetros han sido sumergidos o bien llenados selectivamente con cristales líquidos para así sintonizar la señal interferométrica guiada a través de la fibra. • Infiltración de fibras fotónicas con cristales líquidos colestéricos con especial énfasis en la fase azul (blue phase) de estos materiales. Las moléculas de cristal líquido se autoalinean en volumen por lo que la infiltración de fibras fotónicas con estos cristales líquidos es muy interesante, pues es conocida la dificultad de alinear apropiadamente cristales líquidos dentro de cavidades micrométricas de las fibras fotónicas. • Grabación de redes holográficas de forma selectiva en las cavidades de una fibra fotónica. Estas redes holográficas, llamadas POLICRYPS (POlymer-LIquid CRYstal-Polymer Slices), son redes fabricadas a base de franjas de polímero y cristal líquido alineado perpendicularmente a dichas franjas. Las franjas son a su vez perpendiculares al eje de la fibra como lo puede ser una red de Bragg convencional. El cristal líquido, al estar alineado perpendicularmente a dichos franjas y paralelo al eje de la fibra, se puede conmutar aplicando un campo eléctrico externo, modificando así el índice efectivo de la red. Se puede fabricar por lo tanto una red de Bragg sintonizable en fibra, muy útil en comunicaciones ópticas. • Llenado selectivo de fibras fotónicas con polidimetilsiloxano (PDMS), un polímero de tipo silicona. Si se realiza un llenado selectivo asimétrico se puede inducir birrefringencia en la fibra. El índice de refracción del PDMS tiene una fuerte dependencia térmica, por lo que se puede sintonizar la birrefringencia de la fibra. • Estudio teórico de llenado selectivo de fibras fotónicas con PDMS dopado con nanopartículas de plata de 5, 40 y 80 nm. Estas nanopartículas poseen un pico de absorción en torno a los 450 nm debido a resonancias superficiales localizadas de plasmones (LSPR). La resonancia del plasmon tiene una fuerte dependencia con el índice de refracción del material colindante, y al ser éste PDMS, la variación de índice de refracción se ve amplificada, obteniendo una absorción sintonizable. Se ha propuesto la fabricación de polarizadores sintonizables usando esta técnica. Como ya se ha dicho, previamente a la fabricación ha sido necesaria la protocolización de diversos procedimientos de fabricación de alta complejidad, así como protocolizar el proceso de toma de medidas para optimizar los resultados. Los procedimientos que han requerido la formulación de protocolos específicos han sido los siguientes: • Llenado selectivo de cavidades en una fibra fotónica. Dichas fibras tienen generalmente un diámetro externo de 125 μm, y sus cavidades son de entre 5 y 10 μm de diámetro. Se han desarrollado tres técnicas diferentes para el llenado/bloqueado selectivo, pudiéndose combinar varios protocolos para la optimización del proceso. Las técnicas son las siguientes: o Llenado y bloqueado con un prepolímero. Dicho prepolímero, también llamado adhesivo óptico, está inicialmente en estado líquido y posee una cierta viscosidad. Las cavidades de la fibra fotónica que se desea llenar o bloquear poseen un diámetro diferente al resto, por lo que en el proceso de llenado aparecen dos frentes de llenado dependientes de su diámetro. A mayor diámetro, mayor velocidad de llenado. Polimerizando cuando existe dicha diferencia en los frentes se puede cortar por medio, obteniendo así una fibra parcialmente bloqueada. o Colapsamiento de las cavidades de menor diámetro mediante aplicación de calor. El calor producido por un arco voltaico de una soldadora de fibra estándar fusiona el material exterior de la fibra produciendo el colapsamiento de las cavidades de menor diámetro. En esta técnica también es necesaria una diferencia de diámetros en las cavidades de la fibra. o Bloqueo una a una de las cavidades de la fibra fotónica con adhesivo óptico. Este procedimiento es muy laborioso y requiere mucha precisión. Con este sistema se pueden bloquear las cavidades deseadas de una fibra sin importar su diámetro. • Alineación de una fuente de luz linealmente polarizada con una fibra mantenedora de polarización ya sea PANDA o fotónica. Así mismo también se han alineado entre sí fibras mantenedoras de polarización, para que sus ejes rápidos se fusionen paralelos y así el estado de polarización de la luz guiada se mantenga. • Sistematización de toma de medidas para caracterizar los interferómetros modales. Éstos son altamente sensibles a diversas variables por lo que el proceso de medida es complejo. Se deben aislar variables de forma estrictamente controlada. Aunque todos los dispositivos tienen en común el llenado selectivo de cavidades en una fibra fotónica cada dispositivo tiene sus peculiaridades, que van a ser explicadas a continuación. ABSTRACT The present Thesis has been centered in the design, fabrication and characterization of devices based on photonic crystal fibers selectively filled with liquid crystals, polymers and a mixture of both. All devices are tunable and their work field is optical communications and sensing The handling and splicing of photonic crystal fibers, the selective filling of their holes and the aligning of polarization maintaining fibers are very specific and delicate tasks for which very strict protocols are required. Before the fabrication of devices has therefore been necessary task systematization and creation of manufacturing protocols. Once established we have proceeded to the fabrication and characterization of devices. The fabricated devices are listed below and their peculiarities are detailed one by one: • Intermodal interferometers made with a portion of photonic crystal fiber spliced between two optical communication fiber pigtails, either single mode or PANDA (polarization-maintaining) fiber. These interferometers have been submerged or selectively filled with liquid crystals to tune the interferometric guided signal. • Infiltration of photonic fibers with cholesteric liquid crystals with special emphasis on their blue phase (blue phase). The liquid crystal molecules are self-aligning in volume so the infiltration of photonic fibers with these liquid crystals is very interesting. It is notoriously difficult to properly align liquid crystals within micron cavities such as photonic fibers. • Selectively recording of holographic gratings in the holes of photonic crystal fibers. These holographic gratings, called POLICRYPS (POlymer-LIquid CRYstal-Polymes Slices), are based on walls made of polymer and liquid crystal aligned perpendicular to them. These walls are perpendicular to the axis of the fiber as it can be a conventional Bragg grating. The liquid crystal is aligned perpendicular to the walls and parallel to the fiber axis, and can be switched by applying an external electric field and thus change the effective index of the grating. It is thus possible to manufacture a tunable Bragg grating fiber, useful in optical communications. •Asymmetrically selective filling of photonic crystal fibers with a silicone polymer like called polydimethylsiloxane (PDMS) to induce birefringence in the fiber. The refractive index of PDMS has temperature dependence, so that the birefringence of the fiber can be tuned. • Theoretical study of photonic crystal fibers selectively filled with PDMS doped with silver nanoparticles of 5, 40 and 80 nm. These nanoparticles have an absorption peak around 450 nm due to localized surface plasmon resonances (LSPR). Plasmon resonance has a strong dependence on the refractive index of the adjacent material, and as this is PDMS, the refractive index variation is amplified, obtaining a tunable absorption. Fabrication of tunable polarizers using this technique has been proposed. Before starting the fabrication, it has been necessary to optimize several very delicate procedures and different protocols have been designed. The most delicate procedures are as follows: • Selective filling of holes in a photonic crystal fiber. These fibers generally have an outer diameter of 125 μm, and their holes have a diameter around between 5 and 10 μm. It has been developed three different techniques for filling / selective blocking, and they can be combined for process optimization. The techniques are: o Filling and blocked with a prepolymer. This prepolymer also called optical adhesive is initially in liquid state and has a certain viscosity. The holes of the photonic crystal fiber that are desired to be filled or blocked should have a different diameter, so that in the filling process appear two different fronts depending on the hole diameter. The holes with larger diameter are filled faster. Then the adhesive is polymerized when there is such a difference on the front. A partially blocked fiber is obtained cutting between fronts. o Collapsing of holes of smaller diameter by application of heat. The heat produced by an arc of a standard fusion splicer fuses the outer fiber material producing the collapsing of the cavities of smaller diameter. In this technique also you need a difference of diameters in the fiber holes. o Blocking one by one the holes of photonic crystal fiber with optical adhesive. This procedure is very laborious and requires great precision. This system can block unwanted cavities regardless fiber diameter. • Aligning a linearly polarized light source with a polarization-maintaining fiber (either a PANDA fiber as a photonic crystal fiber). It is needed also an aligning between polarization-maintaining fibers, so that their fast axes parallel merge and that is state of polarization of light guided is maintained. • Systematization of taking measurements to characterize the modal interferometers. These are highly sensitive to several variables so the measurement process is very complicated. Variables must be fixed in a very controlled manner. Although all devices have the common characteristic of being selectively filled PCFs with some kind of material, each one has his own peculiarities, which are explained below.

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate–protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein–protein interaction through the fibronectin type III domains 3–5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein–protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein–protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

The regulators of G protein signaling (RGS) domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gi but not by Gs class α-subunits. All RGS proteins share a conserved 120-amino acid sequence termed the RGS domain. We have demonstrated that the RGS domains of RGS4, RGS10, and GAIP retain GTPase accelerating activity with the Gi class substrates Giα1, Goα, and Gzα in vitro. No regulatory activity of the RGS domains was detected for Gsα. Short deletions within the RGS domain of RGS4 destroyed GTPase activating protein activity and Giα1 substrate binding. Comparable protein–protein interactions between Giα1–GDP–AlF4− and the RGS domain or full-length RGS4 were detected using surface plasmon resonance.

Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56lck, undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56lck, we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48–CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s−1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2–CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of ≈104 M−1 and a dissociation rate of ≥6 s−1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.

Specific molecular recognition of mixed nucleic acid sequences: An aromatic dication that binds in the DNA minor groove as a dimer

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase δ (or ɛ)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36–55 and 196–215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase δ-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase δ-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase δ-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase δ in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.

Syntenin, a PDZ protein that binds syndecan cytoplasmic domains

The syndecans are transmembrane proteoglycans that place structurally heterogeneous heparan sulfate chains at the cell surface and a highly conserved polypeptide in the cytoplasm. Their versatile heparan sulfate moieties support various processes of molecular recognition, signaling, and trafficking. Here we report the identification of a protein that binds to the cytoplasmic domains of the syndecans in yeast two-hybrid screens, surface plasmon resonance experiments, and ligand-overlay assays. This protein, syntenin, contains a tandem repeat of PDZ domains that reacts with the FYA C-terminal amino acid sequence of the syndecans. Recombinant enhanced green fluorescent protein (eGFP)–syntenin fusion proteins decorate the plasmamembrane and intracellular vesicles, where they colocalize and cosegregate with syndecans. Cells that overexpress eGFP–syntenin show numerous cell surface extensions, suggesting effects of syntenin on cytoskeleton–membrane organization. We propose that syntenin may function as an adaptor that couples syndecans to cytoskeletal proteins or cytosolic downstream signal-effectors.

αβ T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10−4 to 10−6M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors

The telomerase enzyme is a potential therapeutic target in many human cancers. A series of potent inhibitors has been designed by computer modeling, which exploit the unique structural features of quadruplex DNA. These 3,6,9-trisubstituted acridine inhibitors are predicted to interact selectively with the human DNA quadruplex structure, as a means of specifically inhibiting the action of human telomerase in extending the length of single-stranded telomeric DNA. The anilino substituent at the 9-position of the acridine chromophore is predicted to lie in a third groove of the quadruplex. Calculated relative binding energies predict enhanced selectivity compared with earlier 3,6-disubstituted compounds, as a result of this substituent. The ranking order of energies is in accord with equilibrium binding constants for quadruplex measured by surface plasmon resonance techniques, which also show reduced duplex binding compared with the disubstituted compounds. The 3,6,9-trisubstututed acridines have potent in vitro inhibitory activity against human telomerase, with EC50 values of up to 60 nM.

A sequential dimerization mechanism for erythropoietin receptor activation.

We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety of in vitro assays. Mutant proteins were expressed in 293s cells and quantified by using an N-terminal epitope tag in conjunction with a surface plasmon resonance assay. Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format binding competition assay and full-length EPO-R in transfected BaF3 cells. Proliferative activity of the mutants was also determined in the BaF3-derived cell line and was correlated with the results from binding assays. Based on the results of these assays, we identified two distinct receptor binding sites on the EPO molecule. We propose that one site, containing residues Arg-150 and Lys-152, binds initially to EPO receptor on the cell surface. A second site, containing Arg-103 and Ser-104 (and possibly Arg-14), is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore, we demonstrate that one EPO mutant (R103A), which has previously been shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.