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Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate–cysteine ligase subunits

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Abstract

Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate–cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser51 phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent.

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Abbreviations

AARE:

Amino acid response element

ATF4:

Activating transcription factor 4

CARE:

CCAAT enhancer-binding protein–activating transcription factor response element

eIF2α:

Eukaryotic initiation factor 2, subunit alpha

EpRE:

Electrophile response element

GCN2:

General control non-derepressible 2, also known as eIF2α kinase 4

GCL:

Glutamate–cysteine ligase

GCLC:

Glutamate–cysteine ligase catalytic subunit

GCLM:

Glutamate–cysteine ligase modifier subunit

GSH:

Glutathione

GSSG:

Glutathione disulfide

MEF:

Murine embryonic fibroblast

Nrf2:

Nuclear factor erythroid 2-related factor 2

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Acknowledgments

The authors gratefully acknowledge Dr. David Ron (New York University School of Medicine, New York, NY, USA) for providing the Gcn2 −/− MEFS; Dr. Randal Kaufman (Sanford Burnham Medical Research Medical Institute, La Jolla, CA, USA) for providing the eIF2α(ala/ala) MEFs; and Dr. M. W. Lieberman (Methodist Hospital Research Institute, Houston, TX) for providing Gclc –/– MEFs. The research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Numbers DK-083473 and Grant DK-056649. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Division of Nutritional Sciences, Cornell University, Ithaca, NY, 14853, USA

    Angelos K. Sikalidis, Kevin M. Mazor, Jeong-In Lee, Heather B. Roman, Lawrence L. Hirschberger & Martha H. Stipanuk

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  1. Angelos K. Sikalidis
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  2. Kevin M. Mazor
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  3. Jeong-In Lee
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  6. Martha H. Stipanuk
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Correspondence to Martha H. Stipanuk.

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Sikalidis, A.K., Mazor, K.M., Lee, JI. et al. Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate–cysteine ligase subunits. Amino Acids 46, 1285–1296 (2014). https://doi.org/10.1007/s00726-014-1687-1

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  • DOI: https://doi.org/10.1007/s00726-014-1687-1

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