Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element.
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.
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112.
Rebecca R
RR
Crawford
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112.
Eugenia T
ET
Prescott
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112.
Charity F
CF
Sylvester
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112.
Ashlee N
AN
Higdon
Department of Biochemistry and Molecular Biology, Shands Cancer Center and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida 32610.
Jixiu
J
Shan
Department of Biochemistry and Molecular Biology, Shands Cancer Center and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida 32610.
Michael S
MS
Kilberg
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112. Electronic address: imungr@lsuhsc.edu.
Imran N
IN
Mungrue
D051700:Activating Transcription Factor 3 / Q000235:genetics / Q000378:metabolism*; D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018384:Oxidative Stress / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D020218:Response Elements / Q000502:physiology*; D017384:Sequence Deletion; D005722:gamma-Glutamylcyclotransferase / Q000096:biosynthesis* / Q000235:genetics
C490449:ATF3 protein, human; C497109:ATF4 protein, human; D051700:Activating Transcription Factor 3; D022762:CCAAT-Enhancer-Binding Proteins; C572776:GGCT protein, human; D012333:RNA, Messenger; D051701:Activating Transcription Factor 4; D005722:gamma-Glutamylcyclotransferase; D005978:Glutathione
ATF3; ATF4; CEBPβ; CHAC1; DNA transcription; DNA-protein interaction; apoptosis; endoplasmic reticulum stress (ER stress); oxidative stress; unfolded protein response (UPR)