Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element.

Abstract

Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the -267 ATF/cAMP response element (CRE) site and a novel -248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease.

• Authors: Rebecca R Crawford, Eugenia T Prescott, Charity F Sylvester, Ashlee N Higdon, Jixiu Shan, Michael S Kilberg, Imran N Mungrue
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 25 Pg. 15878-15891 (Jun 19 2015) ISSN: 1083-351X [Electronic] United States
• PMID: 25931127 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• ATF3 protein, human
• ATF4 protein, human
• Activating Transcription Factor 3
• CCAAT-Enhancer-Binding Proteins
• GGCT protein, human
• RNA, Messenger
• Activating Transcription Factor 4
• gamma-Glutamylcyclotransferase
• Glutathione

Topics

• Activating Transcription Factor 3 (genetics, metabolism)
• Activating Transcription Factor 4 (genetics, metabolism)
• Animals
• Base Sequence
• CCAAT-Enhancer-Binding Proteins (genetics, metabolism)
• Endoplasmic Reticulum Stress (physiology)
• Gene Expression Regulation, Enzymologic (physiology)
• Glutathione (genetics, metabolism)
• HEK293 Cells
• Humans
• Mice
• Oxidative Stress (physiology)
• RNA, Messenger (biosynthesis, genetics)
• Response Elements (physiology)
• Sequence Deletion
• gamma-Glutamylcyclotransferase (biosynthesis, genetics)