title: Alteration of transcriptional and post-transcriptional expression of gamma-glutamylcysteine synthetase by diethyl maleate.
abstract: Gamma-glutamylcysteine synthetase (gamma-GCS), also known as glutamate-cysteine ligase (EC 6.3.2.2), is the rate-limiting enzyme in the synthesis of glutathione (GSH). The gene GLCLC encodes the catalytic subunit while GLCLR encodes the regulatory subunit. Although it has been shown that GLCLC can respond to a variety of stresses by increased transcription, it is not known whether a similar response occurs for GLCLR. Nor is it known whether post-transcriptional regulation of either gene product is altered during stress. The present investigation was undertaken to explore transcriptional and post-transcriptional regulation of GLCLC and GLCLR gene products when HepG2 cells were challenged with the radiation sensitizer diethyl maleate (DEM). Expression of steady-state GLCLC and GLCLR mRNA was enhanced 5-20-fold after DEM challenge. Nuclear run-off assays were performed on unstressed and stressed cells to determine whether the increased expression of GLCLC and GLCLR mRNA was due to altered transcriptional activity of these genes. The DEM treatment increased the transcription rates of both genes 2-5-fold. In unstressed HepG2 cells, the half-life of GLCLC mRNA transcripts was approximately 4 h. In contrast, the half-life of GLCLR transcripts was approximately 8 h. In cells treated with DEM, the half-lives of all transcripts were increased, indicating that message stabilization contributed to the increased expression of gene products. Finally, a PEST algorithm has identified a PEST (proline, glutamate, serine, threonine) motif within the catalytic subunit of gamma-GCS, suggesting that this subunit might exhibit conditional proteolytic regulation. These results imply that regulation of the products of the GLCLC and GLCLR genes may be altered at multiple levels during exposure to stress.
authors.affiliation: Vanderbilt Center for Radiation Oncology, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: K R
authors.initials: KR
authors.lastname: Sekhar
authors.forename: M
authors.initials: M
authors.lastname: Long
authors.forename: J
authors.initials: J
authors.lastname: Long
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Xu
authors.forename: M L
authors.initials: ML
authors.lastname: Summar
authors.forename: M L
authors.initials: ML
authors.lastname: Freeman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D006801:Humans; D008298:Maleates / Q000494:pharmacology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D008298:Maleates; D012333:RNA, Messenger; D005721:Glutamate-Cysteine Ligase; C014476:diethyl maleate