Optimization of glutathione production in Saccharomyces cerevisiae HBSD-W08 using Plackett-Burman and central composite rotatable designs.

BACKGROUND: Glutathione is an important bioactive tripeptide and is widely used in the food, medicine, and cosmetics industries. The aim of this study was to provide an efficient method for producing GSH and to explore its synthesis mechanism. Saccharomyces cerevisiae strain HBSD-W08 was screened for GSH production, and its fermentation medium was optimized using single-factor experiments of the Plackett-Burman and central composite rotatable designs. This method was used to analyze the effects of the presence and concentration of various carbon sources, organic and inorganic nitrogen sources, metal ions, and precursor amino acids on GSH production and catalase, superoxide dismutase, and γ-glutamylcysteine synthetase activity. RESULTS: The three most significant factors affecting GSH production were peptone (optimal concentration [OC]: 2.50 g L- 1), KH2PO4 (OC: 0.13 g L- 1), and glutamic acid (OC: 0.10 g L- 1). GSH productivity of HBSD-W08 was obtained at 3.70 g L- 1 in the optimized medium. The activity of γ-GCS, which is a marker for oxidative stress, was found to be highly positively correlated with GSH production. CONCLUSIONS: This finding revealed an underlying relationship between GSH synthesis and oxidative stress, providing useful information for developing effective GSH fermentation control strategies.

Activation of Nrf2 attenuates carbonyl stress induced by methylglyoxal in human neuroblastoma cells: Increase in GSH levels is a critical event for the detoxification mechanism.

The present study focused on the methylglyoxal (MG) detoxification mechanism in neuroblastoma cells. The involvement of nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway as a defense response against the formation of MG-modified proteins, which is well-known evidence of carbonyl stress, was also examined. We found that MG treatment resulted in accumulation of modified proteins bearing the structure of advanced glycation end products (AGEs) derived from MG in SH-SY5Y cells. This accumulation was suppressed by activation of the Nrf2 pathway prior to MG exposure via pre-treatment with an Nrf2 activator, carnosic acid and CDDO-Im, confirming the involvement of the Nrf2 pathway in MG detoxification. Although pre-treatment with the Nrf2 activator did not affect mRNA levels of GLO1, AKR1B1, and AKR7A2, the expressions of GCL and xCT mRNA, involved in GSH synthesis, were induced prior to increase in GSH levels. Furthermore, we demonstrated that a GSH synthesis inhibitor eliminated the MG detoxification effect derived from pretreatment with the Nrf2 activator. These results indicated that increase in GSH levels, induced by pre-treatment with carnosic acid, promoted the formation of the GLO1 substrate, hemithioacetal, thereby accelerating MG metabolism via the glyoxalase system and suppressing its toxicity. It was, therefore, determined that promotion of GSH synthesis via the Nrf2/Keap1pathway is important in the MG detoxification mechanism against neuronal MG-induced carbonyl stress, and Nrf2 activators contribute to reduction in the accumulation and toxic expression of carbonyl proteins.

A self-amplified ferroptosis nanoagent that inhibits the tumor upstream glutathione synthesis to reverse cancer chemoresistance.

Ferroptosis has recently become an attractive strategy to combat the chemoresistance of cancer cells, but the intracellular ferroptosis defense system greatly challenges the efficient ferroptosis induction. Herein, we report a ferrous metal-organic framework-based nanoagent (FMN) that inhibits the intracellular upstream glutathione synthesis and induces self-amplified ferroptosis of cancer cells, for reversing chemoresistance and boosting chemotherapy. The FMN is loaded with SLC7A11 siRNA (siSLC7A11) and chemotherapeutic doxorubicin (DOX), which shows enhanced tumor cell uptake and retention, thus ensuring the effective DOX delivery and tumor intracellular iron accumulation. Importantly, the FMN simultaneously catalyzes the iron-dependent Fenton reaction and triggers the siSLC7A11-mediated suppression of upstream glutathione synthesis for intracellularly self-amplified ferroptosis, which further inhibits P-glycoprotein activity for DOX retention, and regulates the expression of Bcl-2/Bax to reverse the apoptotic resistance state of tumor cells. The FMN-mediated ferroptosis is also demonstrated in ex vivo patient-derived tumor fragment platform. Consequently, FMN successfully reverses cancer chemoresistance and achieves a highly efficient in vivo therapeutic efficacy in MCF7/ADR tumor-bearing mice. Our study provides a self-amplified ferroptosis strategy via inhibiting intracellular upstream glutathione synthesis, which is effective to reverse cancer chemoresistance.

Inhibition of alanine-serine-cysteine transporter 2-mediated auto-enhanced photodynamic cancer therapy of co-nanoassembly between V-9302 and photosensitizer.

The efficiency of reactive oxygen species (ROS)-based photodynamic therapy (PDT) is far from satisfactory, because cancer cells can adapt to PDT by upregulating glutathione (GSH) levels. The GSH levels in tumor cells are determined based on glutamine availability via alanine-serine-cysteine transporter 2 (ASCT2)-mediated entry into cells. Herein, we develop co-assembled nanoparticles (PPa/V-9302 NPs) of the photosensitizer pyropheophorbide a (PPa) and V-9302 (a known inhibitor of ASCT2) in a 1:1 M ratio using a one-step precipitation method to auto-enhance photodynamic therapy. The computational simulations revealed that PPa and V-9302 could self-assemble through different driving forces, such as π-π stacking, hydrophobic interactions, and ionic bonds. Such PPa/V-9302 NPs could disrupt the intracellular redox homeostasis due to enhanced ROS production via PPa-induced PDT and reduced GSH synthesis via inhibition of the ASCT2-mediated glutamine flux by V-9302. The in vivo assays reveal that PPa/V-9302 NPs could increase the drug accumulation in tumor sites and suppress tumor growth in BALB/c mice bearing mouse breast carcinoma (4 T1) tumor. Our findings provide a new paradigm for the rational design of the PDT-based combinational cancer therapy.

N-cystaminylbiguanide MC001 prevents neuron cell death and alleviates motor deficits in the MPTP-model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), which is highly associated with oxidative stress. Antioxidants are therefore considered as potential therapies in PD treatment. In this study, we examined the neuroprotective effect of a cysteamine-based biguanide N-cystaminylbiguanide (MC001) in the MPTP mouse model of PD. The results showed that MC001 prevented neuron cell death and alleviated motor deficits in the MPTP mouse model of PD. Both in vitro and in vivo data indicated that MC001 may exert its neuroprotective effect by alleviating ROS production, suppressing neuroinflammation, and upregulating BDNF expression. Further mechanistic studies revealed that MC001 promoted GSH synthesis by inducing the expression of Glutamate-cysteine ligase catalytic subunit (Gclc) and enhancing the activity of Glutamate-cysteine ligase (Gcl). Our results suggest that MC001 warrants further investigation as a potential candidate for the treatment of PD.

WRKY74 regulates cadmium tolerance through glutathione-dependent pathway in wheat.

Cadmium (Cd) is a toxic heavy metal to plants and human health. Ascorbate (ASA)-glutathione (GSH) synthesis pathway plays key roles in Cd detoxification, while its molecular regulatory mechanism remains largely unknown, especially in wheat. Here, we found a WRKY transcription factor-TaWRKY74, and its function in wheat Cd stress is not clear in previous studies. The expression levels of TaWRKY74 were significantly induced by Cd stress. Compared to control, the activities of GST, GR, or APX were significantly increased by 1.55-, 1.43-, or 1.75-fold and 1.63-, 2.65-, or 2.30-fold in shoots and roots of transiently TaWRKY74-silenced wheat plants under Cd stress. Similarly, the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), GSH, or Cd were also significantly increased by 2.39- or 1.25-fold, 1.54- or 1.20-fold, and 1.34- or 5.94-fold in shoots or roots in transiently TaWRKY74-silenced wheat plants, while ASA content was decreased by 47.4 or 43.3% in shoots, 10.7 or 6.5% in roots in these silenced wheat plants, respectively. Moreover, the expression levels of GSH, GPX, GR, DHAR, MDHAR, and APX genes, which are involved in ASA-GSH synthesis, were separately induced by 2.42-, 2.16-, 3.28-, 2.08-, 1.92-, and 2.23-fold in shoots, or by 10.69-, 3.33-, 3.26-, 1.81-, 16.53-, and 3.57-fold in roots of the BSMV-VIGS-TaWRKY74-inoculated wheat plants, respectively. However, the expression levels of TaNramp1, TaNramp5, TaHMA2, TaHMA3, TaLCT1, and TaIRT1 metal transporters genes were decreased by 21.2-76.3% (56.6%, 59.2%, 76.3%, 53.6%, 35.8%, and 21.2%) in roots of the BSMV-VIGS-TaWRKY74-inoculated wheat plants. Taken together, our results suggested that TaWRKY74 alleviated Cd toxicity in wheat by affecting the expression of ASA-GSH synthesis genes and suppressing the expression of Cd transporter genes, and further affecting Cd uptake and translocation in wheat plants.

Systemic L-Buthionine -S-R-Sulfoximine Treatment Increases Plasma NGF and Upregulates L-cys/L-cys2 Transporter and γ-Glutamylcysteine Ligase mRNAs Through the NGF/TrkA/Akt/Nrf2 Pathway in the Striatum.

Glutathione (GSH) is the most abundant intracellular antioxidant. GSH depletion leads to oxidative stress and neuronal damage in the central nervous system (CNS). In mice, the acute systemic inhibition of GSH synthesis by L-buthionine-S-R-sulfoximine (BSO) triggers a protective response and a subsequent increase in the CNS GSH content. This response might be modulated by a peripheral increment of circulating nerve growth factor (NGF). NGF is an important activator of antioxidant pathways mediated by tropomyosin-related kinase receptor A (TrkA). Here, we report that peripheral administration of BSO increased plasma NGF levels. Additionally, BSO increased NGF levels and activated the NGF/TrkA/Akt pathway in striatal neurons. Moreover, the response in the striatum included an increased transcription of nrf2, gclm, lat1, eaac1, and xct, all of which are involved in antioxidant responses, and L-cys/L-cys2 and glutamate transporters. Using antibody against NGF confirmed that peripheral NGF activated the NGF/TrkA/Akt/Nrf2 pathway in the striatum and subsequently increased the transcription of gclm, nrf2, lat1, eaac1, and xct. These results provide evidence that the reduction of peripheral GSH pools increases peripheral NGF circulation that orchestrates a neuroprotective response in the CNS, at least in the striatum, through the NGF/TrkA/Akt/Nrf2 pathway.

Influence of GSH synthesis inhibition on temporal distribution of NAD+/NADH during vascular endothelial cells proliferation.

Pathological conditions states such as stroke, diabetes mellitus, hypertension, dyslipidemia are associated with increased levels of free radicals that alter normal function of the vascular endothelium and perturb vascular homeostasis. The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance. Any decrease in tissue or systemic GSH levels under the aforementioned pathologies would enhance oxidative damage to the vascular endothelium. Beside their role as coenzyme that participate in cellular metabolism, pyridine nucleotides serve also as substrate for enzymes involved in DNA repair and longevity. There is scant data on NAD+/NADH kinetics and distribution during human cells proliferation. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C) NAD+ and nuclear NADH kinetics (6 h interval) over 72 h of endothelial cell proliferation. The IHEC cell line was used as a surrogate for human brain micro vascular endothelial cells. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase. When BSO was removed the pattern of nuclear NAD+ resembled that of control group, but nuclear NADH concentrations remained elevated, as in GSH deficient cells (p<0.01). The coincidence of high nuclear NADH and lower nuclear NAD+ with S-phase prolongation are suggestive of CtBP and NAD+-dependent DNA repair enzyme activation under conditions of decreased cellular GSH. These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.