Connecting Protein Conformation and Dynamics with Ligand-Receptor Binding Using Three-Color Förster Resonance Energy Transfer Tracking.

Specific binding between biomolecules, i.e., molecular recognition, controls virtually all biological processes including the interactions between cells and biointerfaces, both natural and synthetic. Such binding often relies on the conformation of biomacromolecules, which can be highly heterogeneous and sensitive to environmental perturbations, and therefore difficult to characterize and control. An approach is demonstrated here that directly connects the binding kinetics and stability of the protein receptor integrin αvß3 to the conformation of the ligand fibronectin (FN), which are believed to control cellular mechanosensing. Specifically, we investigated the influence of surface-adsorbed FN structure and dynamics on αvß3 binding using high-throughput single-molecule three-color Förster resonance energy transfer (FRET) tracking methods. By controlling FN structure and dynamics through tuning surface chemistry, we found that as the conformational and translational dynamics of FN increased, the rate of binding, particularly to folded FN, and stability of the bound FN-αvß3 complex decreased significantly. These findings highlight the importance of the conformational plasticity and accessibility of the arginine-glycine-aspartic acid (RGD) binding site in FN, which, in turn, mediates cell signaling in physiological and synthetic environments.

PET radiopharmaceuticals for imaging integrin expression: tracers in clinical studies and recent developments.

Noninvasive determination of integrin expression has become an interesting approach in nuclear medicine. Since the discovery of the first 18F-labeled cyclic RGD peptide as radiotracer for imaging integrin αvß3 expression in vivo, there have been carried out enormous efforts to develop RGD peptides for PET imaging. Moreover, in recent years, additional integrins, including α5ß1 and αvß6 came into the focus of pharmaceutical radiochemistry. This review will discuss the tracers already evaluated in clinical trials and summarize the preliminary outcome. It will also give an overview on recent developments to further optimize the first-generation compounds such as [18F]Galacto-RGD. This includes recently developed 18F-labeling strategies and also new approaches in 68Ga-complex chemistry. Furthermore, the approaches to develop radiopharmaceuticals targeting integrin α5ß1 and αvß6 will be summarized and discussed.

[Fluorescent fusion proteins with 10th human fibronectin domain].

In the current paper we describe a new type of hybrid molecules including red fluorescent protein mCherry and 10th type III human fibronectin domain (10Fn3) - one of the alternative scaffold proteins which can be used for the construction of antibody mimics with various binding specificity. We have constructed different gene variants encoding for the hybrid fluorescent protein and studied their expression in Escherichia coli cells. It was shown that N-terminal position of mCherry and modification of its N-terminal amino acid sequence promotes efficientbacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construction we have obtained the hybrid fluorescent protein ChIBF, containing alphaVbeta3-integrin binding vari- ant of 10Fn3, and demonstrated the possibility of its utilization for the visualization of alphaVbeta3-integrin at the surface of MDCK epithelial cells by confocal microscopy.

Integrin αvß3-targeted IRDye 800CW near-infrared imaging of glioblastoma.

PURPOSE: Integrin α(v)ß(3) plays an important role in tumor angiogenesis, growth, and metastasis. We have tested a targeted probe to visualize integrin receptor expression in glioblastomas using near-infrared fluorescent (NIRF) imaging. EXPERIMENTAL DESIGN: A transgenic glioblastoma mouse model (RCAS-PDGF-driven/tv-a glioblastoma, which mimics the infiltrative growth pattern of human glioblastomas) and two human orthotopic glioblastoma models (U-87 MG with high integrin ß(3) expression and TS543 with low integrin ß(3) expression) were studied. An integrin-targeting NIRF probe, IRDye 800CW-cyclic-RGD peptide (IRDye 800CW-RGD), was tested by in vivo and ex vivo NIRF imaging. RESULTS: We show that the IRDye 800CW-RGD peptide: (i) specifically binds to integrin receptors; (ii) is selectively localized to glioblastoma tissue with overexpressed integrin receptors and is retained over prolonged periods of time; (iii) is associated with minimal autofluorescence and photobleaching because of imaging at 800 nm; (iv) provides delineation of tumor tissue with high precision because of a high tumor-to-normal brain fluorescence ratio (79.7 ± 6.9, 31.2 ± 2.8, and 16.3 ± 1.3) in the U-87 MG, RCAS-PDGF, and TS543 models, respectively; P < 0.01); and (v) enables fluorescence-guided glioblastoma resection. Importantly, small foci of residual fluorescence were observed after resection was completed using white light imaging alone, and these fluorescent foci were shown to represent residual tumor tissue by histology. CONCLUSIONS: NIRF imaging with the IRDye 800CW-RGD probe provides a simple, rapid, low-cost, nonradioactive, and highly translatable approach for improved intraoperative glioblastoma visualization and resection. It also has the potential to serve as an imaging platform for noninvasive cancer detection and drug efficacy evaluation studies.

Localization of αvß3-like integrin in cultivated larval cells of the mussel Mytilus trossulus during neuronal and muscle differentiation.

Using immunofluorescence phenotyping, the expression of αvß3-like integrin was examined during neuronal and muscle differentiation in cell cultures derived from trochophore larvae of the mussel Mytilus trossulus. We have demonstrated that some mussel cells grown on fibronectin in vitro express the extracellular matrix (ECM) αvß3 integrin-like receptor. At the same time, the distribution of αvß3-like integrin is not ubiquitous, i.e. it depends on the cell type and the time of cultivation. Using immunohistochemical staining, we have found that only in some cells this integrin is co-localized with molluscan neuronal markers, neurotransmitters serotonin (5-HT) or Phe-Met-Arg-Phe-NH(2) neuropeptide (FMRFamide), and also with filament actin but not with paramyosin. Although we have previously shown that an integrin-dependent mechanism is involved in cell adhesion and differentiation of muscle cells of Mytilus, in this study, αvß3-like integrin has not been found to participate in fibronectin adhesion of muscle cells but may be a linking agent between the ECM and the neuron-like cells.

Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines.

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.

Quantifying the specific binding between West Nile virus envelope domain III protein and the cellular receptor alphaVbeta3 integrin.

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.