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1.
Biochem Biophys Rep ; 24: 100834, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33102815

ABSTRACT

The Unfolded Protein Response pathway is a conserved signaling mechanism having important roles in cellular physiology and is perturbed accompanying disease. We previously identified the novel UPR target gene CHAC1, a direct target of ATF4, downstream of PERK-EIF2A and activated by the UPR pathway. CHAC1 enzyme directs catalysis of γ-linked glutamate bonds within specific molecular targets. CHAC1 is the first enzyme characterized that can catalyze intracellular glutathione degradation in eukaryotes, having implications for regulation of oxidative stress. DDIT3 (CHOP) is a terminal UPR transcription factor, regulated by ATF4 and an output promoting cell death signaling. Herein we examine the relationship of CHOP controlling CHAC1 transcription in humans and mice. We note parallel induction of CHOP and CHAC1 in human cells after agonist induced UPR. Expanding upon previous reports, we define transcriptional induction of CHAC1 in humans and mice driven by ATF4 through a synergistic relationship with conserved ATF/CRE and CARE DNA sequences of the CHAC1 promoter. Using this system, we also tested effects of CHOP on CHAC1 transcription, and binding at the CHAC1 ATF/CRE using IM-EMSA. These data indicate a novel inhibitory effect of CHOP on CHAC1 transcription, which was ablated in the absence of the ATF/CRE control element. While direct binding of ATF4 to CHAC1 promoter sequences was confirmed, binding of CHOP to the CHAC1 ATF/CRE was not evident at baseline or after UPR induction. These data reveal CHAC1 as a novel CHOP inhibited target gene, acting through an upstream ATF/CRE motif via an indirect mechanism.

2.
J Biol Chem ; 290(25): 15878-15891, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-25931127

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 ß 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.


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