title: A clickable glutathione approach for identification of protein glutathionylation in response to glucose metabolism.
abstract: Glucose metabolism and mitochondrial function are closely interconnected with cellular redox-homeostasis. Although glucose starvation, which mimics ischemic conditions or insufficient vascularization, is known to perturb redox-homeostasis, global and individual protein glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. In this report, we use our clickable glutathione approach, which forms clickable glutathione (azido-glutathione) by using a mutant of glutathione synthetase (GS M4), for detection and identification of protein glutathionylation in response to glucose starvation. We found that protein glutathionylation is readily induced in HEK293 cells in response to low glucose concentrations when mitochondrial reactive oxygen species (ROS) are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Proteomic and biochemical analysis identified over 1300 proteins, including SMYD2, PP2Cα, and catalase. We further showed that PP2Cα is glutathionylated at C314 in a C-terminal domain, and PP2Cα C314 glutathionylation disrupts the interaction with mGluR3, an important glutamate receptor associated with synaptic plasticity.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
authors.forename: Kusal T G
authors.initials: KT
authors.lastname: Samarasinghe
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
authors.forename: Dhanushka N P
authors.initials: DN
authors.lastname: Munkanatta Godage
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, USA.
authors.forename: Yani
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
authors.forename: Fidelis T
authors.initials: FT
authors.lastname: Ndombera
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
authors.forename: Eranthie
authors.initials: E
authors.lastname: Weerapana
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
authors.forename: Young-Hoon
authors.initials: YH
authors.lastname: Ahn
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005981:Glutathione Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D000071636:Protein Phosphatase 2C / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; C000607456:PPM1A protein, human; D000071636:Protein Phosphatase 2C; D005981:Glutathione Synthase; D005978:Glutathione; D005947:Glucose