Clickable glutathione using tetrazine-alkene bioorthogonal chemistry for detecting protein glutathionylation. 2016

Dilini N Kekulandara, and Kusal T G Samarasinghe, and Dhanushka N P Munkanatta Godage, and Young-Hoon Ahn
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.

Protein glutathionylation is one of the major cysteine oxidative modifications in response to reactive oxygen species (ROS). We recently developed a clickable glutathione approach for detecting glutathionylation by using a glutathione synthetase mutant (GS M4) that synthesizes azido-glutathione (γGlu-Cys-azido-Ala) in situ in cells. In order to demonstrate the versatility of clickable glutathione and to increase the chemical tools for detecting glutathionylation, we sought to develop clickable glutathione that uses tetrazine-alkene bioorthogonal chemistry. Here we report two alkene-containing glycine surrogates (allyl-Gly and allyl-Ser) for the biosynthesis of clickable glutathione and their use for detection, enrichment, and identification of glutathionylated proteins. Our results provide chemical tools (allyl-Gly and allyl-Ser for GS M4) for versatile characterization of protein glutathionylation. In addition, we show that the active site of GS can be tuned to introduce a small size chemical tag on glutathione for exploring glutathione function in cells.

UI MeSH Term Description Entries
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D011499 Protein Processing, Post-Translational Any of various enzymatically catalyzed post-translational modifications of PEPTIDES or PROTEINS in the cell of origin. These modifications include carboxylation; HYDROXYLATION; ACETYLATION; PHOSPHORYLATION; METHYLATION; GLYCOSYLATION; ubiquitination; oxidation; proteolysis; and crosslinking and result in changes in molecular weight and electrophoretic motility. Amino Acid Modification, Post-Translational,Post-Translational Modification,Post-Translational Protein Modification,Posttranslational Modification,Protein Modification, Post-Translational,Amino Acid Modification, Posttranslational,Post-Translational Amino Acid Modification,Post-Translational Modifications,Post-Translational Protein Processing,Posttranslational Amino Acid Modification,Posttranslational Modifications,Posttranslational Protein Processing,Protein Processing, Post Translational,Protein Processing, Posttranslational,Amino Acid Modification, Post Translational,Modification, Post-Translational,Modification, Post-Translational Protein,Modification, Posttranslational,Modifications, Post-Translational,Modifications, Post-Translational Protein,Modifications, Posttranslational,Post Translational Amino Acid Modification,Post Translational Modification,Post Translational Modifications,Post Translational Protein Modification,Post Translational Protein Processing,Post-Translational Protein Modifications,Processing, Post-Translational Protein,Processing, Posttranslational Protein,Protein Modification, Post Translational,Protein Modifications, Post-Translational
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D003545 Cysteine A thiol-containing non-essential amino acid that is oxidized to form CYSTINE. Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D005978 Glutathione A tripeptide with many roles in cells. It conjugates to drugs to make them more soluble for excretion, is a cofactor for some enzymes, is involved in protein disulfide bond rearrangement and reduces peroxides. Reduced Glutathione,gamma-L-Glu-L-Cys-Gly,gamma-L-Glutamyl-L-Cysteinylglycine,Glutathione, Reduced,gamma L Glu L Cys Gly,gamma L Glutamyl L Cysteinylglycine
D005981 Glutathione Synthase One of the enzymes active in the gamma-glutamyl cycle. It catalyzes the synthesis of glutathione from gamma-glutamylcysteine and glycine in the presence of ATP with the formation of ADP and orthophosphate. EC 6.3.2.3. Glutathione Synthetase,Synthase, Glutathione,Synthetase, Glutathione
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000475 Alkenes Unsaturated hydrocarbons of the type Cn-H2n, indicated by the suffix -ene. (Grant & Hackh's Chemical Dictionary, 5th ed, p408) Alkene,Olefin,Olefins,Pentene,Pentenes
D057809 HEK293 Cells A cell line generated from human embryonic kidney cells that were transformed with human adenovirus type 5. 293T Cells,HEK 293 Cell Line,HEK 293 Cells,Human Embryonic Kidney Cell Line 293,Human Kidney Cell Line 293,293 Cell, HEK,293 Cells, HEK,293T Cell,Cell, 293T,Cell, HEK 293,Cell, HEK293,Cells, 293T,Cells, HEK 293,Cells, HEK293,HEK 293 Cell,HEK293 Cell

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