Structural and Functional Analyses of Human ChaC2 in Glutathione Metabolism.

Yen T K Nguyen,Byung Woo Han,Jun Young Jang,Kyu-Won Kim,Kyung Rok Kim,Tam T L Vo,Joon Sung Park

doi:10.3390/biom10010031

Yen T K Nguyen, Byung Woo Han + Show 5 more

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https://doi.org/10.3390/biom10010031

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Journal: Biomolecules	Publication Date: Dec 24, 2019
Citations: 14	License type: CC BY 4.0

Affiliation: Seoul National University, Keimyung University

Abstract

Glutathione (GSH) degradation plays an essential role in GSH homeostasis, which regulates cell survival, especially in cancer cells. Among human GSH degradation enzymes, the ChaC2 enzyme acts on GSH to form 5-l-oxoproline and Cys-Gly specifically in the cytosol. Here, we report the crystal structures of ChaC2 in two different conformations and compare the structural features with other known γ-glutamylcyclotransferase enzymes. The unique flexible loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the conformation of the enzyme and in modulating the enzyme activity. Based on a docking study of GSH to ChaC2 and binding assays, we propose a substrate-binding mode and catalytic mechanism. We also found that overexpression of ChaC2, but not mutants that inhibit activity of ChaC2, significantly promoted breast cancer cell proliferation, suggesting that the GSH degradation by ChaC2 affects the growth of breast cancer cells. Our structural and functional analyses of ChaC2 will contribute to the development of inhibitors for the ChaC family, which could effectively regulate the progression of GSH degradation-related cancers.

Highlights

GSH is a crucial tripeptide (γ-glutamyl-cysteinyl glycine) that participates in diverse cellular functions, including cellular detoxification, redox signaling [1], cell proliferation, and apoptosis [2].Disturbances of GSH homeostasis have been observed in many pathophysiological contexts [3].In particular, an aberrant GSH level is correlated with tumor initiation, progression, and chemotherapeutic resistance [4,5]
We identified the catalytic residues of ChaC2 responsible for the GSH degradation activity and propose that the long flexible loop regulates GSH degradation function
To identify the equivalent catalytic Glu residue of ChaC2, we carefully investigated the active site residues of ChaC2 with other GGCT-fold enzyme family is composed of γ-glutamylcyclotransferase (GGCT) enzymes (Figure 5B)

Summary

Introduction

GSH is a crucial tripeptide (γ-glutamyl-cysteinyl glycine) that participates in diverse cellular functions, including cellular detoxification, redox signaling [1], cell proliferation, and apoptosis [2].Disturbances of GSH homeostasis have been observed in many pathophysiological contexts [3].In particular, an aberrant GSH level is correlated with tumor initiation, progression, and chemotherapeutic resistance [4,5]. GSH is a crucial tripeptide (γ-glutamyl-cysteinyl glycine) that participates in diverse cellular functions, including cellular detoxification, redox signaling [1], cell proliferation, and apoptosis [2]. Disturbances of GSH homeostasis have been observed in many pathophysiological contexts [3]. An aberrant GSH level is correlated with tumor initiation, progression, and chemotherapeutic resistance [4,5]. GSH homeostasis is balanced by GSH biosynthesis, transport and efflux of GSH, GSH-acting enzymes, and GSH degradation [6]. The contribution of GSH biosynthesis has been well studied, the GSH degradation pathway, which involves the initial cleavage of GSH, remains poorly understood. Several enzymes responsible for GSH degradation have been increasingly studied [6]

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