Mammalian proapoptotic factor ChaC1 and its homologues function as γ‐glutamyl cyclotransferases acting specifically on glutathione

Kumar, Akhilesh; Tikoo, Shweta; Maity, Shuvadeep; Sengupta, Shantanu; Sengupta, Sagar; Kaur, Amandeep; Bachhawat, Anand K.

doi:10.1038/embor.2012.156

Cited by 177 publications

(172 citation statements)

References 25 publications

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“…To prevent contamination of endogenous glutathione-degrading enzymes from the yeast lysate, we used a glutathione-degrading enzyme-deficient strain (dug3⌬ ecm38⌬ gcg1⌬ triple mutant) to express the Protein A-tagged ChaC proteins in yeast cells. Consistent with a previous report (18), the beads bound with yeast class I ChaC family protein Gcg1 showed significant GGCT activity (Fig. 2E).…”

Section: Resultssupporting

confidence: 79%

“…As shown in Fig. 5B, the recombinant Gcg1 protein showed significant GGCT activity toward glutathione, as described previously (18). However, we failed to detect GGCT activity of recombinant RipAY, although we could detect robust GGCT activity in RipAY expressed in yeast (Fig.…”

Section: Inoculation Of R Solanacearum Into Eggplant Leaves Causes Amentioning

confidence: 56%

“…Bioinformatics analysis revealed that RipAY contains a ChaC domain, which is a conserved domain found in all phyla examined but whose molecular function was totally unknown when we started our study. Recently, it has been reported that yeast and mammalian ChaC domain-containing proteins exhibit ␥-glutamyl cyclotransferase (GGCT) activity specifically to degrade glutathione (18). We demonstrated that RipAY exhibits robust GGCT activity and significantly decreases intracellular glutathione in yeast.…”

mentioning

confidence: 80%

“…In this regard, we employed a Protein A tag to affinity-purify the proteins using IgG-beads directly from the Protein A-tagged RipAY-expressing yeast lysates and then determined GGCT activity of the beads bound with RipAY protein using a Dug1-coupled assay for ChaC family proteins (18). To prevent contamination of endogenous glutathione-degrading enzymes from the yeast lysate, we used a glutathione-degrading enzyme-deficient strain (dug3⌬ ecm38⌬ gcg1⌬ triple mutant) to express the Protein A-tagged ChaC proteins in yeast cells.…”

Section: Resultsmentioning

confidence: 99%

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RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

Fujiwara

Kawazoe

Ohnishi

et al. 2016

Journal of Biological Chemistry

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The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits ␥-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ϳ30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other ␥-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

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Section: Resultssupporting

confidence: 79%

Section: Inoculation Of R Solanacearum Into Eggplant Leaves Causes Amentioning

confidence: 56%

mentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

Fujiwara

Kawazoe

Ohnishi

et al. 2016

Journal of Biological Chemistry

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“…The higher rate of 5-OP formation from GSH by GGCT2;1 was particularly interesting because, in general, GGCTs accept only g-glutamyl dipeptides as a substrate. To date, the only exception is mammalian ChaC1, which is shown to act on GSH (Kumar et al, 2012). The K m for GSH degradation by GGCT2;1 was 1.93 mM, which was within the range of intracellular GSH concentration as observed in Arabidopsis cells (Fricker et al, 2000).…”

Section: Ggct2;1 Converts G-glutamyl Peptides To 5-opmentioning

confidence: 75%

A -Glutamyl Cyclotransferase Protects Arabidopsis Plants from Heavy Metal Toxicity by Recycling Glutamate to Maintain Glutathione Homeostasis

et al. 2013

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Plants detoxify toxic metals through a GSH-dependent pathway. GSH homeostasis is maintained by the g-glutamyl cycle, which involves GSH synthesis and degradation and the recycling of component amino acids. The enzyme g-glutamyl cyclotransferase (GGCT) is involved in Glu recycling, but the gene(s) encoding GGCT has not been identified in plants. Here, we report that an Arabidopsis thaliana protein with a cation transport regulator-like domain, hereafter referred to as GGCT2;1, functions as g-glutamyl cyclotransferase. Heterologous expression of GGCT2;1 in Saccharomyces cerevisiae produced phenotypes that were consistent with decreased GSH content attributable to either GSH degradation or the diversion of g-glutamyl peptides to produce 5-oxoproline (5-OP). 5-OP levels were further increased by the addition of arsenite and GSH to the medium, indicating that GGCT2;1 participates in the cellular response to arsenic (As) via GSH degradation. Recombinant GGCT2;1 converted both GSH and g-glutamyl Ala to 5-OP in vitro. GGCT2;1 transcripts were upregulated in As-treated Arabidopsis, and ggct2;1 knockout mutants were more tolerant to As and cadmium than the wild type. Overexpression of GGCT2;1 in Arabidopsis resulted in the accumulation of 5-OP. Under As toxicity, the overexpression lines showed minimal changes in de novo Glu synthesis, while the ggct2;1 mutant increased nitrogen assimilation by severalfold, resulting in a very low As/N ratio in tissue. Thus, our results suggest that GGCT2;1 ensures sufficient GSH turnover during abiotic stress by recycling Glu.

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Characterization of a FOXG1:TLE1 transcriptional network in glioblastoma‐initiating cells

et al. 2018

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Glioblastoma (GBM) is the most common and deadly malignant brain cancer of glial cell origin, with a median patient survival of less than 20 months. Transcription factors FOXG1 and TLE1 promote GBM propagation by supporting maintenance of brain tumour‐initiating cells (BTICs) with stem‐like properties. Here, we characterize FOXG1 and TLE1 target genes in GBM patient‐derived BTICs using ChIP‐Seq and RNA‐Seq approaches. These studies identify 150 direct FOXG1 targets, several of which are also TLE1 targets, involved in cell proliferation, differentiation, survival, chemotaxis and angiogenesis. Negative regulators of NOTCH signalling, including CHAC1, are among the transcriptional repression targets of FOXG1:TLE1 complexes, suggesting a crosstalk between FOXG1:TLE1 and NOTCH‐mediated pathways in GBM. These results provide previously unavailable insight into the transcriptional programs underlying the tumour‐promoting functions of FOXG1:TLE1 in GBM.

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Mammalian proapoptotic factor ChaC1 and its homologues function as γ‐glutamyl cyclotransferases acting specifically on glutathione

Cited by 177 publications

References 25 publications

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

A -Glutamyl Cyclotransferase Protects Arabidopsis Plants from Heavy Metal Toxicity by Recycling Glutamate to Maintain Glutathione Homeostasis

Characterization of a FOXG1:TLE1 transcriptional network in glioblastoma‐initiating cells

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