A novel chemical tool to identify glutathione-protein interactions

Nadee Nisanka Jayarathne Matarage Don

doi:10.17918/00010634

The research work described in this thesis is inspired by the emerging non-canonical roles of glutathione (GSH). In addition to maintaining cellular redox homeostasis as a major redox buffer in cells, GSH regulates a variety of other physiological but non-canonical functions, such as regulation of cell apoptosis in eukaryotes, bacterial virulence in prokaryotes and viral morphogenesis in viral strains. Probing these non-canonical roles will unravel novel physiological functions associated with GSH, which would be beneficial for understanding cellular mechanisms and drug development. The first project focused on the identification of GSH-interacting proteins and their physiological importance. A photo-reactive GSH probe (DAZ-G) was developed for labeling and pull-down of GSH-interacting proteins from a mixture of proteins (i.e., cell lysate). The proteomic analysis of cellular proteins derived from HEK293 labeled with DAZ-G identified 52 proteins that showed significant labeling with UV and competition with GSH as potential GSH interacting proteins. Proteomic data were validated using western blot analysis for several hit proteins identified from the proteomics. The direct interaction between these hit proteins and GSH needs to be evaluated along with their physiological importance. This DAZ-G based chemoproteomic approach could be useful for the identification of novel GSH-protein interactions in different organisms such as bacteria and virus. The second project is based on the non-canonical interaction between GSH and the extracellular calcium-sensing receptor (CaSR). The CaSR, abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis and exerts multimodal roles in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we employed the DAZ-G crosslinking approach to analyze ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. Using the DAZ-G probe, we found that caffeine is a novel and potent PAM of CaSR. The effective concentration of caffeine for CaSR activation (<10 [mu]M) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.

A novel chemical tool to identify glutathione-protein interactions

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A novel chemical tool to identify glutathione-protein interactions

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