The dipeptide γ-glutamylcysteine (γ-GC), the first intermediate of glutathione (GSH) synthesis, is considered as a promising drug to reduce or prevent plethora of age-related disorders such as Alzheimer and Parkinson diseases. The unusual γ-linkage between the two constitutive amino acids, namely cysteine and glutamate, renders its chemical synthesis particularly challenging. Herein, we report on the metabolic engineering of the non-conventional yeast Yarrowia lipolytica for efficient γ-GC synthesis. The yeast was first converted into a γ-GC producer by disruption of gene GSH2 encoding GSH synthase and by constitutive expression of GSH1 encoding glutamylcysteine ligase. Subsequently genes involved in cysteine and glutamate anabolism, namely MET4, CYSE, CYSF, and GDH1 were overexpressed with the aim to increase their intracellular availability. With such a strategy, a γ-GC titer of 464 nmol mg-1 protein (93 mg gDCW-1 ) was obtained within 24 h of cell growth.

OBJECTIVE: Fusarium graminearum is a destructive fungal pathogen that causes Fusarium head blight (FHB) on a wide range of cereal crops. To control fungal diseases, it is essential to comprehend the pathogenic mechanisms that enable fungi to overcome host defenses during infection. Pathogens require an oxidative stress response to overcome host-derived oxidative stress. Here, we identify the underlying mechanisms of the Fgbzip007-mediated oxidative stress response in F. graminearum. ChIP-seq and subsequent genetic analyses revealed that the role of glutathione in pathogenesis is not dependent on antioxidant functions in F. graminearum. Altogether, this study establishes a comprehensive framework for the Fgbzip007 regulon on pathogenicity and oxidative stress responses, offering a new perspective on the role of glutathione in pathogenicity.

Type 2 diabetes mellitus (T2DM), a complex metabolism disease, which was characterized by metabolic disorders including hyperglycemia, has become a major health problem due to the increasing prevalence worldwide. γ-glutamylcysteine (γ-GC) as an immediate precursor of glutathione (GSH) was originally used for the treatment of sepsis, inflammation bowel disease, and senescence. Here, we evaluated the capacity of γ-GC on diabetes-related metabolic parameters in db/db mice and insulin resistance (IR) amelioration in cells induced by palmitic acid (PA). Our data suggested that γ-GC treatment decreased body weight, reduced adipose tissue size, ameliorated ectopic fat deposition in liver, increased the GSH content in liver, improved glucose control and other diabetes-related metabolic parameters in vivo. Moreover, in vitro experiments showed that γ-GC could maintain the balance of free fatty acids (FFAs) and glucose uptake through regulating the translocation of CD36 and GLUT4 from cytoplasm to plasma membrane. Furthermore, our finding also provided evidence that γ-GC could activate Akt not only via adenylate cyclase (AC)/cAMP/PI3K signaling pathway, but also via IGF-1R/IRS1/PI3K signaling pathway to improve IR and hepatic steatosis. Blocking either of two signaling pathways could not activate Akt activation induced by γ-GC. This unique characteristic ensures the important role of γ-GC in glucose metabolism. Collectively, these results suggested that γ-GC could serve as a candidate dipeptide for the treatment of T2DM and related chronic diabetic complications via activating AC and IGF-1R/IRS1/PI3K/Akt signaling pathways to regulate CD36 and GLUT4 trafficking.

Environmental exposures often produce reactive electrophiles in vivo, leading to oxidative stress, which plays a major role in carcinogenesis. These electrophiles frequently form adducts with human albumin, which can be measured to assess in vivo oxidative stress. Here, we aimed to examine the associations between circulatory albumin adducts and acute myeloid leukemia (AML), the most common adult myeloid leukemia that showed consistent associations with environmental exposures. We conducted a nested case-control study of 52 incident AML cases and 103 controls matched on age, sex and race within two prospective cohorts: the CLUE and PLCO studies. We measured 42 untargeted albumin adducts in prediagnostic samples using liquid chromatography-high-resolution mass spectrometry. Circulatory albumin adducts were associated with AML in conditional logistic regression models. For instance, higher levels of Cys34 disulfide adduct of the S-γ-glutamylcysteine, a precursor of the essential antioxidant, glutathione were associated with a lower risk of AML (odds ratios [95% confidence intervals]) for the 1st, 2nd and 3rd tertiles were 1.0, 0.65 (0.31-1.36) and 0.31 (0.12-0.80), respectively (P-trend = .01). These associations were largely driven by effects present among cases diagnosed at or above the median follow-up time of 5.5 years. In conclusion, applying a novel approach to characterize exposures in the prediagnostic samples, we found evidence supporting the notion that oxidative stress may play a role in the pathogenesis of AML. Our findings offer insight into AML etiology and may be relevant in identifying novel therapeutic targets.

OBJECTIVE: To explore the molecular mechanism of γ-glutamylcysteine (γ-GC) in response to inflammation in vivo and in vitro on regulating the polarization of macrophages.
METHODS: The expressions of gene or protein were assessed by qPCR and Western blot assays, respectively. Cell viability was investigated by CCK-8 assay. Eight-week-old male BALB/c mice were established to examine the therapeutic effects of γ-GC in vivo. The release of TNF-α and IL-4 was determined by ELISA assay. Macrophages polarization was identified by flow cytometry assay.
RESULTS: Our data showed that γ-GC treatment significantly improved the survival, weight loss, and colon tissue damage of IBD mice. Furthermore, we established M1- and M2-polarized macrophages, respectively, and our findings provided evidence that γ-GC switched M1/M2-polarized macrophages through activating AMPK/SIRT1 axis and inhibiting inflammation-related signaling pathway.
CONCLUSIONS: Collectively, both in vivo and in vitro experiments suggested that γ-GC has the potential to become a promising novel therapeutic dipeptide for the treatment of IBD, which provide new ideas for the treatment of inflammatory diseases in the future.

Rheumatoid arthritis (RA) is an essential cause of labor loss and disability for people worldwide. Acanthopanax senticosus polysaccharide (ASPS) is one of the most important active components from A. senticosus, which exhibits various pharmacological activities such as antioxidation and immunomodulation. However, no studies have reported the application of ASPS in treating RA. This study aims to investigate the therapeutic effect of ASPS on RA and reveal its underlying mechanism. The potential therapeutic effect of ASPS against RA is initially verified in this study using the collagen-induced arthritis model. Moreover, the protective benefits of ASPS are transmitted through the fecal microbiota and blocked by simultaneous antibiotic cocktail treatment, indicating that gut microbiota may be correlated with ASPS. The 16S rRNA sequencing using feces samples and untargeted UPLC-MS metabolomics using serum samples further reveal that ASPS reprograms the arthritic progression triggered dysbiosis, enhances the expression of γ-glutamylcysteine (GGC) synthetase, and enriches the serum concentration of GGC. Furthermore, metabolites GGC is found to be able to effectively interrupt NLRP3 inflammasome activation via inhibiting ASC nucleation and therefore attenuate inflammatory arthritis. Taken together, this work highlights ASPS\'s therapeutic potential against RA, which mainly exhibits its effects via modulating gut microbiota and regulating GGC production.

Ferroptosis is a non-apoptotic form of cell death driven by iron-dependent lipid peroxidation. Recent evidence indicates that inhibiting ferroptosis could alleviate cerebral ischemia/reperfusion (CIR) injury. γ-glutamylcysteine (γ-GC), an intermediate of glutathione (GSH) synthesis, can upregulate GSH in brains. GSH is the co-factor of glutathione peroxidase 4 (GPX4), which is the negative regulator of ferroptosis. In this study, we explored the effect of γ-GC on CIR-induced neuronal ferroptosis and brain injury. We found that γ-GC significantly reduced the volume of cerebral infarction, decreased the loss of neurons and alleviated neurological dysfunction induced by CIR in rats. Further observation showed that γ-GC inhibited the CIR-caused rupture of the neuronal mitochondrial outer membrane and the disappearance of cristae, and decreased Fe2+ deposition and lipid peroxidation in rat cerebral cortices. Meanwhile, γ-GC altered the expression of some ferroptosis-related proteins in rat brains. Mechanistically, γ-GC increased the expression of GSH synthetase (GSS) for GSH synthesis via protein kinase C (PKC)ε-mediated activation of nuclear factor erythroid 2-related factor (Nrf2). Our findings suggest that γ-GC not only serves as a raw material but also increases the GSS expression for GSH synthesis against CIR-induced lipid peroxidation and ferroptosis. Our study strongly suggests that γ-GC has potential for treating CIR injury.

BACKGROUND: Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices.
METHODS: After synthesizing [34S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota.
RESULTS: Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r2 > 0.997), recovery (90.9%-110%) and stability (90.0%-100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0-66.7% reduction with antibiotics).
CONCLUSIONS: A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.

Alcohol abuse is a major cause of alcoholic liver disease (ALD) and can result in fibrosis and cirrhosis. γ-glutamylcysteine (γ-GC) is a precursor of glutathione (GSH) with antioxidant and anti-inflammatory properties. Our research aimed to explore the protective impact of γ-GC on ALD and its potential mechanisms of efficiency in vitro and in vivo. L02 cells were pretreated with γ-GC (20, 40, and 80 μM) for 2 h and exposed to ethanol for 24 h. Cell viability, apoptosis, oxidative stress, and inflammatory levels were measured. The expression of protein cleaved caspase-3 and cleaved PARP and flow cytometry results indicated that γ-GC decreases apoptosis on L02 cells after ethanol treatment. Moreover, γ-GC also attenuated oxidative stress and mitochondrial damage in hepatocytes caused by ethanol via increasing cellular GSH, SOD activity, and mitochondrial membrane potential. In vivo experiments, γ-GC effectively reduced the AST, ALT, and TG levels in mice. The inflammation of ALD was alleviated by γ-GC both in vivo and in vitro. Additionally, histopathological examination demonstrated that γ-GC treatment lessened lipid droplet formation and inflammatory damage. In conclusion, these results showed that γ-GC has anti-inflammatory and anti-apoptotic effects on ALD because it could help hepatocytes maintain sufficient GSH levels to combat the excess reactive oxygen species (ROS) generated during ethanol metabolism. PRACTICAL APPLICATIONS: Alcohol intake is the fifth highest risk factor for premature death and disability among all risk variables. However, few medicines are both safe and effective for the treatment of ALD. As a direct precursor of GSH, γ-GC has a broad variety of potential antioxidant and anti-inflammatory applications for the treatment of numerous medical conditions. In conclusion, these results showed that γ-GC could protect cells from ALD via suppressing oxidative stress, alleviating inflammation, and preventing apoptosis.

Alzheimer\'s disease (AD) is the most prevalent neurogenerative disease, characterized by progressive memory loss and cognitive deficits. Intracellular neurofibrillary tangles (NFTs) and amyloid-β (Aβ)-formed neuritic plaques are major pathological features of AD. Aβ evokes activation of microglia to release inflammatory mediators and ROS to induce neurotoxicity, leading to neurodegeneration. γ-Glutamylcysteine (γ-GC), an intermediate dipeptide of the GSH-synthesis pathway with anti-inflammatory and anti-oxidative properties, represents a relatively unexplored option for AD treatment. In the present study, we investigated the anti-inflammatory effect of γ-GC on Aβ oligomer (AβO)-induced neuroinflammation and the associated molecular mechanism in microglia. The results showed that γ-GC reduced AβO-induced release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO), and the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). γ-GC decreased ROS and MDA production and increased the GSH level, GSH/GSSG ratio, and SOD activity in AβO-treated microglia. Mechanistically, γ-GC inhibited activation of nuclear factor kappa B (NF-κB), and upregulated the nuclear receptor-related 1 (Nurr1) protein expression to suppress the transcriptional effect of NF-κB on the inflammatory genes. Besides, γ-GC suppressed the AβO-induced neuroinflammation in mice. These findings suggested that γ-GC might represent a potential therapeutic agent for anti-neuroinflammation.