title: Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity.
abstract: The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H(2)O(2)-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.
authors.affiliation: Department of Pharmaceutical Sciences, Graduate Program in Toxicology, School of Pharmacy, University of Colorado Denver, Aurora, CO 80045, USA.
authors.forename: Donald S
authors.initials: DS
authors.lastname: Backos
authors.forename: Chad N
authors.initials: CN
authors.lastname: Brocker
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Franklin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000096:biosynthesis*; D051379:Mice; D009154:Mutation; D016877:Oxidants / Q000633:toxicity*; D021122:Protein Subunits; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D016877:Oxidants; D021122:Protein Subunits; D014157:Transcription Factors; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione