Proximity Ligation Assay to Study Protein–protein Interactions of Proteins on two different Cells

Abstract

Protein-protein interactions (PPI) by homo-, hetero- or oligo-merization in the cellular environment regulate cellular processes. PPI can be inhibited by antibodies, small molecules or peptides, and this inhibition has therapeutic value. A recently developed method, the proximity ligation assay (PLA), provides detection of PPI in the cellular environment. However, most applications using this assay are for proteins expressed in the same cell. We employ PLA for the first time to study PPI of cell surface proteins on two different cells. Inhibition of PPI using a peptide inhibitor is also quantified using this assay; PLA is used to detect PPI of CD2 and CD58 between Jurkat cells (T cells) and human fibroblast-like synoviocyte-rheumatoid arthritis cells that are important in the immune response in the autoimmune disease rheumatoid arthritis. This assay provides direct evidence of inhibition of PPI of two proteins on different cell surfaces.

Human papillomavirus type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response

“…This assay can in situ identify, validate and characterize interactions in native proteins in their correct tissue/cell context under near natural/physiological conditions, without requiring protein content extraction from tissue or the need to over-express the target proteins. Conversely, as also observed by other authors, since co-IP needs high concentrations of both target proteins to generate effective signals and is often dependent on antibodies affinity [ 42 , 43 , 52 – 58 ], it can’t be the right way to see protein interactions in our 3D models where cellular density and consequently target proteins are not so highly expressed.…”

“…Since PLA technology allows the in situ detection, with sub-cellular resolution and molecular biology precision (0–40 nm), of protein-protein interactions within tissues [ 42 , 43 , 52 – 58 ], we revealed E6 and E7 antigens binding with 53BP1 by circularized, ligated and amplified complementary and fluorescent labeled oligonucleotides bounded antibodies in the 3D model. Two negative controls were performed (data not shown) by: i ) omission of the primary antibodies and ii ) single antibody incubation.…”

Increased talin–vinculin spatial proximities in livers in response to spotted fever group rickettsial and Ebola virus infections

“…A practical step toward unraveling the complex molecular relationship, temporal and spatial, of protein-to-protein physical interaction is essential for accomplishing a comprehensive knowledge of the functional outcomes [82]. Since detection of protein-protein proximity has been developed to probe the proximal PPI in native environment [16,83,84], PLA is being more and more widely used in tissues and cells [22,23]. In a typical PLA experiment, two antibodies are used to recognize two proteins of interest.…”

Single-Cell Analysis of Multiple Steps of Dynamic NF-κB Regulation in Interleukin-1α-Triggered Tumor Cells Using Proximity Ligation Assays

“…Therefore, PLAs are increasingly used for the analysis of signaling in tumor cells to improve the accuracy of diagnosis [14,46]. These assays can be also performed with non-adherent cells such as lymphomas and they can even visualize interactions of proteins contained on the surface of two different cells [47,48]. The constant development of PLA protocols together with progress in the generation of high quality antibodies, standardized protocols, automated picture analysis, and a pipeline of bioinformatics analysis tools will enable a quantitative, reliable, and comparable analysis of dynamic signaling events in single cells and further improve molecular diagnostics of tumors.…”