Degradation of glutathione and glutathione conjugates in plants.

Glutathione (GSH) is a ubiquitous, abundant, and indispensable thiol for plants that participates in various biological processes, such as scavenging reactive oxygen species, redox signaling, storage and transport of sulfur, detoxification of harmful substances, and metabolism of several compounds. Therefore knowledge of GSH metabolism is essential for plant science. Nevertheless, GSH degradation has been insufficiently elucidated, and this has hampered our understanding of plant life. Over the last five decades, the γ-glutamyl cycle has been dominant in GSH studies, and the exoenzyme γ-glutamyl transpeptidase has been regarded as the major GSH degradation enzyme. However, recent studies have shown that GSH is degraded in cells by cytosolic enzymes such as γ-glutamyl cyclotransferase or γ-glutamyl peptidase. Meanwhile, a portion of GSH is degraded after conjugation with other molecules, which has also been found to be carried out by vacuolar γ-glutamyl transpeptidase, γ-glutamyl peptidase, or phytochelatin synthase. These findings highlight the need to re-assess previous assumptions concerning the γ-glutamyl cycle, and a novel overview of the plant GSH degradation pathway is essential. This review aims to build a foundation for future studies by summarizing current understanding of GSH/glutathione conjugate degradation.

MIA3 promotes the degradation of GSH (glutathione) by binding to CHAC1, thereby promoting the progression of hepatocellular carcinoma.

MIA3 (melanoma inhibitory active protein 3)/TANGO1 (Golgi transporter component protein) plays an important role in the initiation, development, and metabolism of cancer. We aimed to explore the role and underlying molecular mechanisms of MIA3/TANGO1 in the growth and migration of hepatoma cells. According to the analysis of The Cancer Genome Atlas (TCGA) database, MIA3 is expressed at higher levels in hepatocellular carcinoma (HCC) tissues than in normal tissues. Real-time quantitative polymerase chain reaction (qRT-PCR), immunohistochemistry, and western blotting were used to detect mRNA and protein expression in HCC tissues and cells. The in vitro function of MIA3 in HCC cells was evaluated using Cell Counting Kit-8 (CCK-8), colony formation, cell migration and invasion, and flow cytometry assays. Hep-G2 cells with MIA3 overexpression were subjected to RNA-seq, and the downstream target gene CHAC1 (glutathione-specific γ-glutamyl cyclotransferase 1) was selected according to the results of the volcano map of gene enrichment. The relationship between MIA3 and CHAC1 was revealed by coimmunoprecipitation and confocal microscopy. MIA3 expression was upregulated in HCC organizations and HCC samples in the TCGA dataset. Knocking out MIA3 inhibited the proliferation, migration, and invasion of Hep-G2 cells and promoted the apoptosis of Hep-G2 cells. Overexpression of MIA3 in Huh7 cells promoted the proliferation, migration, and invasion and suppressed the apoptosis of Huh7 cells. Overexpression of MIA3 promoted the expression of CHAC1 and the degradation of glutathione (GSH), thereby promoting the growth and metastasis of HCC cells. Knocking out MIA3 inhibited the expression of CHAC1 and slowed the degradation of glutathione, thereby inhibiting the growth and metastasis of HCC cells. MIA3 further promotes the growth, metastasis, and invasion of hepatoma cells by binding to the CHAC1 protein and promoting GSH degradation.

Characterization of glutathione-specific gamma glutamyl cyclotransferase (ChaC) in Bombyx mori.

Glutathione (GSH) contributes to redox maintenance and detoxification of various xenobiotic and endogenous substances. γ-glutamyl cyclotransferase (ChaC) is involved in GSH degradation. However, the molecular mechanism underlying GSH degradation in silkworms (Bombyx mori) remains unknown. Silkworms are lepidopteran insects that are considered to be an agricultural pest model. We aimed to examine the metabolic mechanism underlying GSH degradation mediated by B. mori ChaC and successfully identified a novel ChaC gene in silkworms (herein, bmChaC). The amino acid sequence and phylogenetic tree revealed that bmChaC was closely related to mammalian ChaC2. We overexpressed recombinant bmChaC in Escherichia coli, and the purified bmChaC showed specific activity toward GSH. Additionally, we examined the degradation of GSH to 5-oxoproline and cysteinyl glycine via liquid chromatography-tandem mass spectrometry. Quantitative real-time polymerase chain reaction revealed that bmChaC mRNA expression was observed in various tissues. Our results suggest that bmChaC participates in tissue protection via GSH homeostasis. This study provides new insights into the activities of ChaC and the underlying molecular mechanisms that can aid the development of insecticides to control agricultural pests.

Host-specific activation of a pathogen effector Aave_4606 from Acidovorax citrulli, the causal agent for bacterial fruit blotch.

RipAY, an effector protein from the plant bacterial pathogen Ralstonia solanacearum, exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the host cellular glutathione (GSH) when stimulated by host eukaryotic-type thioredoxins (Trxs). Aave_4606 from Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbit plants, shows significant homology to RipAY. Based on its homology, it was predicted that the GGCT activity of Aave_4606 is also stimulated by host Trxs. The GGCT activity of a recombinant Aave_4606 protein was investigated in the presence of various Trxs, such as yeast (ScTrx1), Arabidopsis thaliana (AtTrx-h1, AtTrx-h2, AtTrx-h3, and AtTrx-h5), or watermelon (Cla022460/ClTrx). Unlike RipAY, the GGCT activity of Aave_4606 is stimulated only by AtTrx-h1, AtTrx-h3, AtTrx-h5 and ClTrx from a watermelon, the primary host of A. citrulli, but not by ScTrx1, AtTrx-h2. Interestingly, GGCT activity of Aave_4606 is more efficiently stimulated by AtTrx-h1 and ClTrx than AtTrx-h5. These results suggested that Aave_4606 recognizes host-specific Trxs, which specifically activates the GGCT activity of Aave_4606 to decrease the host cellular GSH. These findings provide new insights into that effector is one of the host-range determinants for pathogenic bacteria via its host-dependent activation.

Ggct (γ-glutamyl cyclotransferase) plays an important role in erythrocyte antioxidant defense and red blood cell survival.

The expression of GGCT (γ-glutamyl cyclotransferase) is upregulated in various human cancers. γ-glutamyl cyclotransferase enzyme activity was originally purified from human red blood cells (RBCs), but the physiological function of GGCT in RBCs is still not clear. Here we reported that Ggct deletion in mice leads to splenomegaly and progressive anaemia phenotypes, due to elevated oxidative damage and the shortened life span of Ggct-/- RBCs. Ggct-/- RBCs have increased reactive oxygen species (ROS), and are more sensitive to H2 O2 -induced damage compared to control RBCs. Glutathione (GSH) and GSH synthesis precursor l-cysteine are decreased in Ggct-/- RBCs. Our study suggests a critical function of Ggct in RBC redox balance and life span maintenance through regulating GSH metabolism.

Arabidopsis γ-glutamylcyclotransferase affects glutathione content and root system architecture during sulfur starvation.

γ-Glutamylcyclotransferase initiates glutathione degradation to component amino acids l-glutamate, l-cysteine and l-glycine. The enzyme is encoded by three genes in Arabidopsis thaliana, one of which (GGCT2;1) is transcriptionally upregulated by starvation for the essential macronutrient sulfur (S). Regulation by S-starvation suggests that GGCT2;1 mobilizes l-cysteine from glutathione when there is insufficient sulfate for de novo l-cysteine synthesis. The response of wild-type seedlings to S-starvation was compared to ggct2;1 null mutants. S-starvation causes glutathione depletion in S-starved wild-type seedlings, but higher glutathione is maintained in the primary root tip than in other seedling tissues. Although GGCT2;1 is induced throughout seedlings, its expression is concentrated in the primary root tip where it activates the γ-glutamyl cycle. S-starved wild-type plants also produce longer primary roots, and lateral root growth is suppressed. While glutathione is also rapidly depleted in ggct2;1 null seedlings, much higher glutathione is maintained in the primary root tip compared to the wild-type. S-starved ggct2;1 primary roots grow longer than the wild-type, and lateral root growth is not suppressed. These results point to a role for GGCT2;1 in S-starvation-response changes to root system architecture through activity of the γ-glutamyl cycle in the primary root tip. l-Cysteine mobilization from glutathione is not solely a function of GGCT2;1.

γ-Glutamyl cyclotransferase contributes to tumor progression in high grade serous ovarian cancer by regulating epithelial-mesenchymal transition via activating PI3K/AKT/mTOR pathway.

OBJECTIVE: High grade serous ovarian cancer (HGSC) remains one of the most lethal malignancies in females. We previously reported that γ-glutamyl cyclotransferase (GGCT) was significantly upregulated in serous ovarian cancer. The current study was aimed to explore the function and underlying mechanism of GGCT in HGSC. METHODS: GGCT expression was assessed by immunohistochemistry in 128 HGSC patients. Stable cell lines with GGCT gene overexpression or knockdown were established to investigate the function of GGCT in HGSC in vitro and in vivo. RESULTS: GGCT is highly upregulated in HGSC tissues and associated with FIGO stage, lymph node metastasis and ascitic fluid volume. High expression of GGCT is associated with poor survival in HGSC patients. The Harrell's c-indexes of the prognostic models for overall survival and progression-free survival prediction were 0.758 and 0.726, respectively. GGCT knockdown suppresses proliferation, clone formation, migration, and invasion of tumor cells in vitro while forced GGCT overexpression presents opposite results. Furthermore, GGCT silencing inhibits tumor growth and spread in vivo. Epithelial-mesenchymal transition (EMT) and PI3K/AKT/mTOR signaling pathway are suppressed in GGCT silenced cells and enhanced in GGCT overexpressed cells. Inactivation of PI3K/AKT/mTOR signaling pathway in GGCT overexpressed cells induces EMT inhibition. CONCLUSIONS: Our data reveals an important role of GGCT in regulating EMT and progression of HGSC, providing a valuable prognostic marker and potential target for treatment of HGSC patients.

Signaling pathways upregulating glutathione­specific γ­glutamylcyclotransferase 1 by 3­(5'­hydroxymethyl­2'­furyl)­1­benzylindazole.

Glutathione­specific γ­glutamylcyclotransferase 1 (CHAC1), is an unfolded protein response­induced gene. Although it has been previously reported that CHAC1 transcription is regulated by activating transcription factor (ATF) 4, ATF3 and CCAAT/enhancer­binding protein ß (C/EBPß), the signaling pathways that regulate CHAC1 are largely unknown. It was revealed that 3­(5'­hydroxymethyl­2'­furyl)­1­benzylindazole (YC­1; PubChem ID: 5712), a nitric oxide­independent activator of soluble guanylyl cyclase (sGC), increases CHAC1 levels in cultured human kidney proximal tubular cells (HK­2). Therefore, in the present study, the signaling pathways that induce CHAC1 by YC­1 were investigated in HK­2 cells. YC­1 induced CHAC1 expression in a dose­ and time­dependent manner. KT5823, an inhibitor of cGMP­dependent protein kinase (PKG), partially inhibited CHAC1 upregulation, indicating that the sGC­cGMP­PKG pathway participates in CHAC1 regulation. These results also suggested that other signaling pathways are involved in the regulation of CHAC1. Since antibody array analysis showed the activation of p38, mTOR and Akt, the involvement of these factors was further investigated. Although LY294002 and KU0063794 (inhibitors of Akt and mTOR, respectively) inhibited YC­1­induced CHAC1 expression, SB203580 (an inhibitor of p38) did not. These results indicated that CHAC1 is regulated by the Akt­mTOR pathway. In addition, YC­1 induced endoplasmic reticulum (ER) stress, a regulator of CHAC1 induction. These findings suggested that CHAC1 is regulated by YC­1 through the sGC­cGMP­PKG, Akt­mTOR and ER stress pathways. The present study demonstrated that CHAC1 induction reduced the intracellular glutathione concentration, indicating that CHAC1 plays an important role in intracellular redox homeostasis in tubular cells.

γ-Glutamyl cyclotransferase contributes to endometrial carcinoma malignant progression and upregulation of PD-L1 expression during activation of epithelial-mesenchymal transition.

Recent increases in the incidence of endometrial carcinoma represent a significant risk to women's health. We found that γ-glutamyl cyclotransferase (GGCT) was significantly up-regulated in endometrial carcinoma tissues and cells, which suggested that it may be a potential target for treatment of endometrial carcinoma. Furthermore, the impact of GGCT on proliferation, migration, and invasion of endometrial carcinoma has been demonstrated in vitro and in vivo using GGCT silencing and overexpression techniques. In addition, the epithelial-mesenchymal transition (EMT) was significantly inhibited in response to GGCT knockdown, which indicated that GGCT may contribute endometrial carcinoma malignancy during activation of the EMT. We also found that GGCT regulated PD-L1 expression during EMT activation. Furthermore, co-culture of endometrial carcinoma cells with CD8+ T lymphocytes showed that downregulation of PD-L1 expression following GGCT knockdown contributed to the killing activity of CD8+ T lymphocytes on endometrial carcinoma cells. In conclusion, our study showed that GGCT contributed to malignant progression and upregulation of PD-L1 expression of endometrial carcinoma, and may be a potential target for treatment of endometrial carcinoma.

Association of epigenetic alterations in the human C7orf24 gene with the aberrant gene expression in malignant cells.

Human chromosome 7 open reading frame 24 (C7orf24)/γ-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics.