2Institute of Pathology, University of Regensburg, Regensburg, Germany
A.R. and J.S. contributed equally to this article.
Analyses of protein interactions are fundamental for the investigation of molecular mechanisms responsible for cellular processes and diseases, as well as for drug discovery in the pharmaceutical industry. The present study details the development of a fluorescence polarization assay using melanoma inhibitory activity (MIA) protein–binding compounds and studies of the binding properties of this protein. Since they are dependent on the the lifetime of the fluorescent label, currently available fluorescence polarization assays can only determine interactions with either high– or low–molecular weight interaction partners. Our new approach eliminates this limitation by immobilizing a known binding partner of MIA protein to a well plate and by labeling the target protein using luminescent transition metal labels such as Ru(bpy)3 for binding studies with both high– and low–molecular weight interaction partners. Due to the use of a functionalized surface, we termed our concept heterogeneous transition metal–based fluorescence polarization (HTFP) assay. The assay's independence from the molecular weight of potential binding partners should make the technique amenable to investigations on subjects as diverse as multimerization, interactions with pharmacophores, or binding affinity determination.
Protein interactions play a fundamental role in many biochemical processes like signal transduction, immune reaction, cell cycle control, differentiation, and protein folding. The search for potent and selective inhibitors for specific protein-protein binding events is essential in pharmaceutical drug design and various techniques have been established for probing these interactions. One commonly used screening method is an in-solution fluorescence polarization (FP) assay; in this technique, the rotational mobility of a fluorescently labeled entity is assessed in order to provide information about whether it is in a bound or unbound state. Clearly, this method is dependent upon there being a significant difference in molecular weight between the unbound fluorescently labeled entity versus when it is part of a binding complex. Here, we describe a high-throughput FP-based screening assay that is compatible with both high– and low–molecular weight interaction partners.
We have termed our method the heterogeneous transition metal–based fluorescence polarization (HTFP) assay. Compared with organic fluorophores, luminescent transition metal complexes have a number of advantages, such as a large Stokes shift, high photostability, and the option to be used in time-gated measurements. These time-gated measurements offer the possibility of multiple labeling using transition metals with different lifetimes, despite possible spectral overlap and the elimination of auto fluorescence of biological material. Although luminescent transition metal complexes have been used in FP immunoassays (1,2,3,4,5,6,7,8,9,10,11,12,13), these reagents are not feasible for use in binding investigations of low–molecular weight compounds in solution (e.g., in drug candidate screening) due to the dependence of fluorescence polarization on molecular weight and fluorescence lifetime. Therefore, instead of performing the assay in free solution, we have anchored one of the binding factors to the surface of a well in a multiwell plate. This surface-bound factor is then incubated with its fluorescently labeled protein partner and other compounds being tested for binding activity. In the absence of any competitive inhibitor, the protein is tethered to the well's surface and has limited ability to rotate. However, in the presence of a compound that can compete with the binding between the protein and its immobilized binding partner, the protein is freed into solution, and the resulting increase in its rotational diffusion can be easily detected by FP measurements.
We demonstrate the HTFP assay in a screen of melanoma inhibitory activity (MIA) protein inhibitors. MIA protein, an 11 -kDa molecule produced under physiological conditions by cartilage, is strongly expressed and secreted by malignant melanoma cells, but is not expressed in melanocytes (14,15). Since MIA protein expression level in vivo directly correlates with progressive malignancy of melanocytic tumors, it serves as a reliable clinical tumor marker to detect and monitor metastatic diseases (1,16,17). Recent data describe a direct interaction of MIA protein with the cell adhesion receptors integrin α4β1 and integrin α5β1, and with extracellular matrix molecules, including fibronectin (18,19). By modulating integrin activity and masking matrix structures, MIA protein mediates detachment of melanoma cells, resulting in enhanced invasive and migratory potential that ultimately contributes significantly to the formation of metastasis (20). To prevent metastasis, it is desirable to find substances that specifically bind to MIA protein and thereby reduce MIA protein–induced effects. Here, MIA protein and known binding partners were used to develop the HTFP assay as a high-throughput method to identify and characterize protein interactions.Materials and methods Cell lines and cell culture conditions
The melanoma cell line Mel Im, established from a human metastatic tumor sample (provided by Judith Johnson, University of Munich, Germany), was used in Boyden Chamber invasion experiments. Cells were maintained in DMEM (PAA Laboratories GmbH, Pasching, Austria) supplemented with penicillin (400 U/mL), streptomycin (50 µg/mL), L-glutamine (300 µg/mL), and 10% FCS (Pan Biotech GmbH, Germany) and split in a 1:5 ratio every 3 days.Boyden Chamber invasion assay
Invasion assays were performed in Boyden Chambers containing polycarbonate filters with 8-µm pore size (Neuro Probe, Gaithersburg, MD, USA) essentially as described previously (21). Filters were coated with Matrigel, a commercially available reconstituted basement membrane (diluted 1:3 in water; BD Biosciences, San Jose, CA, USA). The lower compartment was filled with fibroblast-conditioned medium used as a chemoattractant. Mel Im melanoma cells were harvested by trypsinization for 2 min, resuspended in DMEM without FCS at a density of 2.5 × 104 cells/mL, and placed in the upper compartment of the chamber. Except for the control experiment with untreated cells and experiments where cells were only treated with the peptide, MIA protein or Ru(bpy)3-labeled MIA protein, respectively, was added to the cell suspension at a final concentration of 200 ng/mL. Peptide AR54 (sequence NSLLVSFQPPRAR) was used at a final concentration of 1 µM. After incubation at 37°C for 4 h, filters were removed. Cells adhering to the lower surface of the filter were fixed, stained, and counted. Experiments were carried out in triplicate and repeated at least three times.