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

Journal Article · Mon Feb 15 00:00:00 EST 2010 · Toxicology and Applied Pharmacology

DOI:https://doi.org/10.1016/j.taap.2009.11.010· OSTI ID:21344863

Backos, Donald S; Brocker, Chad N ^[1]

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1. Department of Pharmaceutical Sciences, Graduate Program in Toxicology, School of Pharmacy, University of Colorado Denver, Aurora, CO 80045 (United States)

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{sub 2}O{sub 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.

• OSTI ID: 21344863
• Journal Information: Toxicology and Applied Pharmacology, Vol. 243, Issue 1; Other Information: DOI: 10.1016/j.taap.2009.11.010; PII: S0041-008X(09)00476-1; Copyright (c) 2009 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; ISSN 0041-008X
• Country of Publication: United States
• Language: English

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60 APPLIED LIFE SCIENCES
AIDS VIRUS
ANTIOXIDANTS
APOPTOSIS
BIOSYNTHESIS
CAPACITY
CYSTEINE
DOSES
GLUTATHIONE
INJURIES
LIGASES
MUTANTS
OXIDATION
TOXICITY
AMINO ACIDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DISEASES
DRUGS
ENZYMES
MICROORGANISMS
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ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PEPTIDES
POLYPEPTIDES
PROTEINS
RADIOPROTECTIVE SUBSTANCES
RESPONSE MODIFYING FACTORS
SYNTHESIS
THIOLS
VIRUSES