Advanced technology for comprehensive analysis of membrane-protein-extracellular interactions.

(Left). Schematic diagram of AirID linked to the gene at the antigen binding site of an antibody. FabID is a genetic fusion of AirID into the antigen binding site (right). Schematic diagram of membrane protein exPPI analysis using FabID. The molecule marked B in pink represents biotin, and proteins near the ear ID are biotinylated labeled. Credit: Kohdai Yamada, Sawasaki Tatsuya (Ehime University)

Many proteins within the body form complexes with other proteins, determining the fate of cells. Therefore, the analysis of protein-protein interactions (PPI) is an important process for understanding the biological function of target proteins.

Membrane protein, which accounts for more than 30% Plays an important role in cell functions. Very are known to form complexes to exert their functions, making membrane proteins essential for understanding the specificity of PPIs. . However, the development of technologies to analyze PPIs, particularly extracellular protein–protein interactions (exPPIs) of living cells, has been delayed.

Recently, proximity labeling methods that label proteins proximally for large-scale PPI analysis have gained attention. However, proximal protein labeling methods targeting the extracellular region of membrane proteins primarily involve PPI analysis using molecules with cellular toxicity, leading to the development of living cell targeting systems. A search prompt appears.

The Proteoscience Center of Eiheim University has independently developed proximity-dependent Biotin labeling enzyme AirIDwhich biotinylates adjacent protein lysine residues.

There have been several studies using affinity-dependent biotin-labeling enzymes for XPPI analysis, but these included genetically modified proteins with conformations significantly different from their original structure, which It is not clear to what extent the results of the analysis reflect the actual interactions.

To accurately understand the interactions occurring at the cell membrane of living cells, it was necessary to develop a technology that could directly target proteins expressed by cells for EXPPI analysis. Therefore, the research group began this study on the premise that it would be possible to analyze exPPI by creating a molecule (FabID) that fuses AirID to the antigen recognition site of an antibody that recognizes the extracellular domain of membrane proteins. Is. Here is the research. published In the journal Nature Communications.

Advanced technology for comprehensive analysis of membrane-protein-extracellular interactions.

Green represents subcellular localization of EGFR and red represents biotinylated labeled protein. Higher red color in the lower row indicates that the protein is more biotinylated. The cells were epithelial cancer cells (A431 cells). Credit: Kohdai Yamada, Sawasaki Tatsuya (Ehime University)

The membrane protein analyzed for exPPI was the epidermal growth factor receptor EGFR, a protein known to be a cancer gene located on the cell membrane. When FabID and biotin were added to epithelial-like carcinoma-derived cells (A431 cells) expressing EGFR (hereafter referred to as epithelial cancer cells), it was confirmed at the cell culture level that EGFR FR can be biotinylated using FabID.

By combining biotin labeling with FabID and Developed at the University of Tokushima for the analysis of biotinylated proteins, the researchers successfully identified several novel EGFR-interacting proteins. The identified proteins have the potential to become new drug targets.

EGFR transmits signals to cells by binding to a ligand called EGF. It is known that when EGF binds to EGFR, different proteins bind to the intracellular domain of EGFR, forming a protein complex.

Currently, EGFR tyrosine kinase inhibitors, which are widely used as cancer treatments, are thought to bind to EGFR and block the formation of protein complexes mediated by the EGFR intracellular domain. exerts its therapeutic effects. However, ligand-dependent and drug-dependent exPPI changes of EGFR occur when EGFR binds to EGF (ligand) or when EGFR tyrosine kinase inhibitors (drugs) act.

Researchers observed ligand-dependent and drug-dependent exPPI changes of EGFR in cultured cells using FabID. As a result, they believe they are the first in the world to find that exPPI changes dynamically when ligands and drugs bind to EGFR.

Advanced technology for comprehensive analysis of membrane-protein-extracellular interactions.

A) Heat map diagram showing biotinylation changes in DMSO (without ligand and drug addition), EGF (ligand) and EGF+Gefitinib (ligand + drug) treated zones. The cells were epithelial cancer cells (A431 cells). Proteins in black letters are known EGFR-interacting proteins, blue letters are EGFR-interacting proteins newly identified in the present study. Credit: Kohdai Yamada, Sawasaki Tatsuya (Ehime University)

The ripple effect

Membrane proteins are used by almost all organisms to transfer information in and out of cells. Therefore, analyzing EXPPI of membrane proteins is expected to contribute directly to the development of receptor biology. Furthermore, since the majority of commercial drugs act by targeting membrane proteins, membrane proteins are known to be important drug targets.

However, finding new membrane protein drug targets is difficult, and this has been a major problem in the pharmaceutical industry. The FabID technology developed in this study not only enables EXPPI analysis using living cells, but can also be used to identify new drug targets.

Furthermore, FabID has been found to capture ligand-dependent and drug-dependent exPPI changes that have not been analyzed by conventional methods. In the future, the identification of new drug targets by exPPI analysis using FabID and the detailed analysis of membrane protein exPPI changes when binding to commercial drugs are expected to play an important role in the development of commercial drugs. will do

This research was conducted as a joint research project of Ehime University Proteoscience Center, Tokushima University Institute of Advanced Medical Sciences, Tokyo Pharmaceutical University School of Life Sciences, Tohoku University Graduate School of Medicine Department of Molecular Pharmacology. and Department of Bioscience, Nagahama Institute of Bioscience and Technology.

More information:
Kohdai Yamada et al, Proximity Extracellular Protein-Protein Interaction Analysis of EGFR Using an AirID-conjugated Fragment of Antigen Binding, Nature Communications (2023). DOI: 10.1038/s41467-023-43931-7

Provided by
Ehime University

Reference: New technology allows comprehensive analysis of extracellular interactions of membrane proteins (2024, February 22) Accessed 22 February 2024 at https://phys.org/news/2024-02-technology-comprehensive-analysis-membrane-protein. Retrieved from html

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