Chemoproteomics is a technique where the cellular proteome is labelled and identified with chemical probes. It has attracted attention as a robust approach to interrogating the functional states of cellular proteins. Recent works by the Nakamura and Hamachi groups reported the proximal labelling of selective proteins by combining singlet oxygen-generating protein ligands and nitrogen-containing nucleophilic probes. Inspired by these works, the research group led by Motonari Uesugi postulated that it is possible to capture and analyze blue-light-oxidized intracellular proteins with nitrogen-containing nucleophilic probes at proteome levels.
The team started with screening for nonaromatic, low-molecular-weight alkyne amine probes. Ideal chemical probes suitable for proteome analysis need to have great cell permeability, reactivity, and selectivity. In an in vitro testing environment, HeLa cells were irradiated with 450 nm blue light for 10 minutes. The nitrogen-containing nucleophilic probes and flavin mononucleotide (a photosensitizer that generates ROS) were sequentially added. Labelled proteins were then visualized by a click reaction with the fluorescent dye TAMRA azide. Fluorescence scanning of SDS-PAGE gels showed that probes 1 and 2 had comparable levels of protein labelling. Similarly, probe 1 outperformed the other two probes in a similar in cellulo testing. It was concluded that probe 1 is the most suitable candidate for further proteome analysis.
Figure 1: Proteome labeling with amine probes under blue light irradiation. Probe 1 (propargylamine was selected for further investigations).
Identification of the blue-light-oxidized proteins is the next crucial step in the research. The team treated both HeLa and B16F10 cells with probe 1 under blue-light irradiation for 10 minutes. The cells were then lysed and reacted with Dde-biotin azide. The biotinylated proteins were isolated by avidin agarose, eluted by hydrazine, and analyzed by LC-MS/MS for label-free quantification. To the researcher’s surprise, cell surface proteins were more readily oxidized than proteins located in other major organelles (such as mitochondria). Specifically, the integrin family members are very sensitive to blue-light irradiation.
Using the data obtained from these two tests, the research team was able to locate a specific integrin protein, Integrin β1 (ITGB1) from HeLa, B16F10, and iHCEC cells. ITGB1 is a cell-surface protein that forms heterodimers with integrin α family members to control cell adhesion, migration, and survival. Upon binding to the extracellular matrix (ECM), ITGB1 undergoes a conformational change from bent to extended forms, thereby clustering intracellular adaptor proteins to induce phosphorylation of focal adhesion kinase. Under the light-stressed condition, singlet oxygen oxidizes ITGB1, thus blunting its ability to stimulate the ECM-dependent downstream signalling. As predicted, researchers found a 70 percent decrease in the activated forms of ITGB1 in the exposure to blue light. The result also confirmed that probe 1 detects the photooxidation of histidines and tyrosines in ITHB1. However, how the oxidations of histidine and/or tyrosine oxidations affect the function of ITGB1 remains unclear.
Figure 2: Structural representation of ITGB1 protein and analysis of ITGB1 function after blue-light irradiation.
In summary, the Uesugi Group identified a light-sensitive protein that can be used to facilitate the study of photodamaged proteins. While the current detection is only limited to side chains of tyrosine and histidine residues, further development of oxidation-dependent probes selective for other amino acids can broaden this field.
The finding of this research has been published on Journal of American Chemical Society: Toh, K.; Nishio, K.; Nakagawa, R.; Egoshi, S.; Abo, M.; Perron, A.; Sato, S.; Okumura, N.; Koizumi, N.; Dodo, K.; Sodeoka, M.; Uesugi, M. Chemoproteomic Identification of Blue-Light-Damaged Proteins. J. Am. Chem. Soc. 2022. ASAP. https://doi.org/10.1021/jacs.2c07180
Comentarios