Proteomics of purified lamellocytes from Drosophila melanogaster HopTum-l identifies new membrane proteins and networks involved in their functions.

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.

Preclinical Development and First-in-Human Imaging of the Integrin αvß6 with [18F]αvß6-Binding Peptide in Metastatic Carcinoma.

PURPOSE: The study was undertaken to develop and evaluate the potential of an integrin αvß6-binding peptide (αvß6-BP) for noninvasive imaging of a diverse range of malignancies with PET. EXPERIMENTAL DESIGN: The peptide αvß6-BP was prepared on solid phase and radiolabeled with 4-[18F]fluorobenzoic acid. In vitro testing included ELISA, serum stability, and cell binding studies using paired αvß6-expressing and αvß6-null cell lines. In vivo evaluation (PET/CT, biodistribution, and autoradiography) was performed in a mouse model bearing the same paired αvß6-expressing and αvß6-null cell xenografts. A first-in-human PET/CT imaging study was performed in patients with metastatic lung, colon, breast, or pancreatic cancer. RESULTS: [18F]αvß6-BP displayed excellent affinity and selectivity for the integrin αvß6 in vitro [IC50(αvß6) = 1.2 nmol/L vs IC50(αvß3) >10 µmol/L] in addition to rapid target-specific cell binding and internalization (72.5% ± 0.9% binding and 52.5% ± 1.8%, respectively). Favorable tumor affinity and selectivity were retained in the mouse model and excretion of unbound [18F]αvß6-BP was rapid, primarily via the kidneys. In patients, [18F]αvß6-BP was well tolerated without noticeable adverse side effects. PET images showed significant uptake of [18F]αvß6-BP in both the primary lesion and metastases, including metastasis to brain, bone, liver, and lung. CONCLUSIONS: The clinical impact of [18F]αvß6-BP PET imaging demonstrated in this first-in-human study is immediate for a broad spectrum of malignancies.

Isolation of integrin-based adhesion complexes.

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry.

Elevación de la troponina cardiaca I en corredoras de raids de aventura / Cardiac troponin I increases in female adventure racers

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Protein expression and purification of integrin I domains and IgSF ligands for crystallography.

Cell adhesion depends on combinational expression and interactions of a large number of adhesion molecules at cell-to-cell or cell-to-matrix contact sites. Integrins and their immunoglobulin superfamily (IgSF) ligands represent foremost classes of cell adhesion molecules in immune system. Structural study is critical for a better understanding of the interactions between integrins and their IgSF ligands. Here we describe protocols for protein expression of integrin αL I domain and its IgSF ligand ICAM-5 D1D2 fragment for crystallography.

Electron microscopic imaging of integrin.

Rotary-shadowed samples often used for electron microscopy do not preserve native integrin conformations. Negatively stained integrins - or, more desirably, unstained integrins in a cryo-condition - are now being used with sophisticated imaging techniques. Additionally, a single-particle analysis (SPA) of integrins is advanced by the recent determination of several crystal structures of integrins. Nevertheless the conformational flexibility of integrins limits the ability of SPA to image physiologic conformations. To solve this problem, we apply electron tomography to purified integrin, thereby obtaining high-quality three-dimensional (3-D) images that fit well to the atomic structures. We have also taken typical SPA approaches to obtain a 3-D reconstruction of integrin, using conditions that favor the bent conformation.

Analyzing integrin-dependent adhesion.

In this unit, methods for the analysis of integrin-dependent adhesion are described. Two major types of assays are commonly used for this analysis. The first are cell adhesion assays (as described in UNIT 9.1). A key application of this type of assay is to identify which integrin(s) mediate cell-substrate interactions; a comprehensive list of antibodies suitable for this purpose is detailed. The second are solid-phase assays in which purified integrins and integrin ligands are used. These assays can be used, e.g., to measure apparent affinities of integrins for different ligands and IC(50) values of pharmacological inhibitors.

Gene expression in SK-Mel-28 human melanoma cells treated with thesnake venom jararhagin

Alternative approaches to improve the treatment of advanced melanomas are highly needed.The disintegrin domain of metalloproteinases binds integrin receptors on tumor cells,blocking migration, invasion, and metastatization. Previous studies showed that jararhagin,from the Bothrops jararaca snake venom, induces changes in the morphology and viability ofSK-Mel-28 human melanoma cells, and decreases the number of metastases in mice injected with pre-treated cells. The purpose of this study was to evaluate the molecular effects ofjararhagin on SK-Mel-28 cells and fibroblasts, concerning the expression of integrins, cadherins, caspases, and TP53 genes. Sub-toxic doses of jararhagin were administered to confluent cells. RT-PCR was performed following extraction of total RNA. Jararhagin treatmentsinduced similar morphological alterations in both normal and tumor cells, with higher IC50 values for fibroblasts. Integrin genes were downregulated in untreated cells,except for ITGA6a,b, ITGAv, and ITGB3 which were highly expressed in SK-Mel-28. The integrin expression profiles were not affected by the toxin. However, jararhagin 30 ng/mlupregulated genes TP53, CDKN1A, CDKN2A, CASP3, CASP5, CASP6, CASP8, and E-CDH in SKMel-28, and genes ITGB6, ITGB7, CASP3, TP53, and CDKN1B in fibroblasts. Appropriate jararhagin concentration can have apoptotic and suppressant effects on SK-Mel-28 cells, ratherthan on fibroblasts, and can be used to develop potential anti-cancer drugs.

La matriz extracelular: de la mecánica molecular al microambiente tumoral (parte II) / The extracellular matrix: From the molecular mechanics to the tumoral microenvironment (part II)

IntroducciónLa matriz extracelular (MEC) representa una red tridimensional que engloba todos los órganos, tejidos y células del organismo. Constituye un filtro biofísico de protección, nutrición e inervación celular y el terreno para la respuesta inmune, angiogénesis, fibrosis y regeneración tisular. También representa el medio de transmisión de fuerzas mecánicas a la membrana basal, que a través de las integrinas soporta el sistema de tensegridad y activa los mecanismos epigenéticos celulares.Método y resultadosLa revisión de la literatura y actualización del tema muestran como la alteración de la MEC supone la pérdida de su función de filtro eficaz, nutrición, eliminación, denervación celular, pérdida de la capacidad de regeneración y cicatrización y alteración de la transmisión mecánica o mecanotransducción. También la pérdida del sustrato para una correcta respuesta inmune ante agentes infecciosos, tumorales y tóxicos.Método y resultadosEsta segunda parte de revisión de la MEC considera los tumores como tejidos funcionales conectados y dependientes del microambiente. El microambiente tumoral, constituido por la MEC, células del estroma y la propia respuesta inmune son determinantes de la morfología y clasificación tumoral, agresividad clínica, pronóstico y respuesta al tratamiento del tumor.ConclusiónTanto en condiciones fisiológicas como patológicas, la comunicación recíproca entre células del estroma y el parénquima dirige la expresión génica. La capacidad oncogénica del estroma procede tanto de los fibroblastos asociados al tumor como de la celularidad de la respuesta inmune y la alteración de la tensegridad por la MEC. La transición epitelio-mesenquimal es el cambio que transforma una célula normal o «benigna» en «maligna»...(AU)

IntroductionThe extracellular matrix (ECM) is a three-dimensional network that envelopes all the organs, tissues and cells of the body. A biophysical filter that provides protection, nutrition and cell innervation, it is the site for the immune response, angiogenesis, fibrosis and tissue regeneration. It is also the transport medium for mechanical forces to the basal membrane through integrins that support the tensegrity system, activating cellular epigenetic mechanisms.Method and resultsThe review of the literature shows how the disruption of the ECM leads to a functional loss of nutrition, elimination, cell innervation, regenerative capacity and wound healing as well as alterations in mechanical transduction. This loss also disrupts the immune response to pathogens, tumour cells and toxins.Method and resultsThe second part of our revision of the ECM considers tumours as interconnected, functional tissues dependant on their microenvironment. The tumoural microenvironment, which is comprised of the ECM, stromal cells and the immune response, determines the morphology of the tumour and its classification as well as its clinical aggressivity, prognosis and response to treatment.ConclusionsBoth in physiological and pathological conditions, the reciprocal communication between the cells of the stroma and the parenchyma direct the genetic expression. The onocogenic capacity of the stroma depends not only on the fibroblasts associated with the tumour but also on the cellularity of the immune response and the alteration in the tensegrity model of the ECM. The epithelial-mesenchymal transition is the change which transforms a normal, or benign, cell into a malignant one. The “pseudomesenchymal” cytoskeleton provides the properties of migration, invasion and dissemination, and vice-versa: the malignant phenotype is reversible by correction of the key factors that create the tumoural microenvironment(AU)

Purification, analysis, and crystal structure of integrins.

Integrins are large modular cell-surface receptors that regulate almost every aspect of cellular function through bidirectional signals transmitted across the lipid bilayer. Regulation of integrin activity is accomplished by complex and still incompletely understood biochemical pathways that modify integrin ligand binding, clustering, trafficking, and signaling functions. The dynamic tertiary and quaternary changes required to channel some of these activities have hampered, until recently, the crystal structure determination of these heterodimeric receptors. In this chapter, we review the methods used to purify and characterize these proteins biophysically and functionally, and to derive their three-dimensional structures.

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers.

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4-0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.

IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.

Recent work from several laboratories has demonstrated that proteolytic mechanisms significantly contribute to the molecular interplay between Streptococcus pyogenes, an important human pathogen, and its host. Here we describe the identification, purification and characterization of a novel extracellular cysteine proteinase produced by S.pyogenes. This enzyme, designated IdeS for Immunoglobulin G-degrading enzyme of S.pyogenes, is distinct from the well-characterized streptococcal cysteine proteinase, SpeB, and cleaves human IgG in the hinge region with a high degree of specificity. Thus, other human proteins, including immunoglobulins M, A, D and E, are not degraded by IdeS. The enzyme efficiently cleaves IgG antibodies bound to streptococcal surface structures, thereby inhibiting the killing of S.pyogenes by phagocytic cells. This and additional observations on the distribution and expression of the ideS gene indicate that IdeS represents a novel and significant bacterial virulence determinant, and a potential therapeutic target.

Analyzing integrin-dependent adhesion.

This unit describes methods for the analysis of integrin-ligand binding in both cell-based assays and solid-phase assays. A major application of cell adhesion assays is in investigating whether a certain cell type can adhere to a specific adhesive substrate, and, if so, which receptors are involved. Particularly if the substrate is a matrix component (e.g., fibronectin), members of the integrin family are likely to play a dominant role in adhesion. Procedures are described here for assessing which integrins are involved in this process. A detailed analysis of ligand recognition by individual integrins can be performed using a solid-phase receptor-ligand binding assay. The unit also contains support protocols for integrin purification and coupling of antibodies to Sepharose for use in this purification, as well as for biotinylation of integrin ligands to be used in the solid-phase assay.

A monoclonal antibody to platelet type III collagen-binding protein (TIIICBP) binds to blood and vascular cells, and inhibits platelet vessel-wall interactions.

TIIICBP is a new platelet receptor involved in platelet-type III collagen and platelet-subendothelium interactions. This receptor is composed of a doublet of 72-68 kDa proteins. In this study, the major protein (68 kDa) was purified and used to produce monoclonal antibodies. One of these antibodies, 7F4, binds to platelets as confirmed by flow cytometry. 7F4 inhibited platelet contact, spreading and aggregation induced by type III collagen. Under flow conditions, 7F4 prevented platelet interactions with type III collagen, endothelial cell matrix and the KOGEOGPK type II collagen octapeptide: the specific sequence recognized by TIIICBP. On the other hand, 7F4 had no effect on platelet-type I collagen interactions. TIIICBP was also detected on lymphocytes, granulocytes and monocytes. TIIICBP was expressed on endothelial cells and fibroblasts but not on smooth-muscle cells. These results show that TIIICBP is expressed on several cell types and participates in cell adhesion to the subendothelium.

Identification of a collagen-binding protein from Necator americanus by using a cDNA-expression phage display library.

A phage display library was made starting from a cDNA library from the hematophagous human parasite Necator americanus. The cDNA library was transferred by polymerase chain reaction (PCR) cloning into phage display vectors (phagemids), using specially designed primers such that proteins would be expressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vectors described by Jespers et al. (1995). Electroporation of the ligation mixtures into electrocompetent Escherichia coli TGI cells yielded 3 x 10(8) pG6A, 1.9 x 10(8) pG6B, and 1 x 10(8) pG6C transfectants for N. americanus. The final libraries consisted of a mix of equal numbers of insert-containing phages from the A, B, and C libraries. Selection of phages for binding to human collagen was performed. Four rounds of panning on human collagens I and III resulted in a significant enrichment of collagen-binding phages from the N. americanus libraries. PCR analysis revealed various insert lengths; however, sequence determination indicated that all phages contained the same protein, albeit with different poly-A tail lengths. The encoded protein itself is a 135-amino acid protein (15 kDa), with no apparent homology to any other known protein. Next the protein was recloned into E. coli using the pET-15b-vector. Upon isopropyl-1-thio-beta-D-galactopyranoside induction, the recombinant protein, rNecH1, could be recovered by urea treatment from inclusion bodies. The rNecH1 protein binds to different collagens: human I > rat I > human III = calf skin I in a specific, dose-dependent, and saturable manner.

Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen.

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.

Vitronectin regulates Sonic hedgehog activity during cerebellum development through CREB phosphorylation.

During development of the cerebellum, Sonic hedgehog (SHH) is expressed in migrating and settled Purkinje neurons and is directly responsible for proliferation of granule cell precursors in the external germinal layer. We have previously demonstrated that SHH interacts with vitronectin in the differentiation of spinal motor neurons. Here, we analysed whether similar interactions between SHH and extracellular matrix glycoproteins regulate subsequent steps of granule cell development. Laminins and their integrin receptor subunit alpha6 accumulate in the outer most external germinal layer where proliferation of granule cell precursors is maximal. Consistent with this expression pattern, laminin significantly increases SHH-induced proliferation in primary cultures of cerebellar granule cells. Vitronectin and its integrin receptor subunits alpha(v) are expressed in the inner part of the external germinal layer where granule cell precursors exit the cell cycle and commence differentiation. In cultures, vitronectin is able to overcome SHH-induced proliferation, thus allowing granule cell differentiation. Our studies indicate that the pathway in granule cell precursors responsible for the conversion of a proliferative SHH-mediated response to a differentiation signal depends on CREB. Vitronectin stimulates phosphorylation of cyclic-AMP responsive element-binding protein (CREB), and over-expression of CREB is sufficient to induce granule cell differentiation in the presence of SHH. Taken together, these data suggest that granule neuron differentiation is regulated by the vitronectin-induced phosphorylation of CREB, a critical event that terminates SHH-mediated proliferation and permits the differentiation program to proceed in these cells.