title: Metabolic synthesis of clickable glutathione for chemoselective detection of glutathionylation.
abstract: Glutathionylation involves reversible protein cysteine modification that regulates the function of numerous proteins in response to redox stimuli, thereby altering cellular processes. Herein we developed a selective and versatile approach to identifying glutathionylation by using a mutant of glutathione synthetase (GS). GS wild-type catalyzes coupling of γGlu-Cys to Gly to form glutathione. We generated a GS mutant that catalyzes azido-Ala in place of Gly with high catalytic efficiency and selectivity. Transfection of this GS mutant (F152A/S151G) and incubation of azido-Ala in cells efficiently afford the azide-containing glutathione derivative, γGlu-Cys-azido-Ala. Upon H2O2 treatment, clickable glutathione allowed for selective and sensitive detection of glutathionylated proteins by Western blotting or fluorescence after click reaction with biotin-alkyne or rhodamine-alkyne. This approach affords the efficient metabolic tagging of intracellular glutathione with small clickable functionality, providing a versatile handle for characterizing glutathionylation.
authors: Department of Chemistry, Wayne State University , Detroit, Michigan 48202, United States.
authors: Kusal T G
authors: KT
authors: Samarasinghe
authors: Dhanushka N P
authors: DN
authors: Munkanatta Godage
authors: Garrett C
authors: GC
authors: VanHecke
authors: Young-Hoon
authors: YH
authors: Ahn
mesh_terms: D055162:Biocatalysis; D057930:Click Chemistry; D005978:Glutathione / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005981:Glutathione Synthase / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D005981:Glutathione Synthase; D005978:Glutathione