MGST3 regulates BACE1 protein translation and amyloidogenesis by controlling the RGS4-mediated AKT signaling pathway.

Microsomal glutathione transferase 3 (MGST3) regulates eicosanoid and glutathione metabolism. These processes are associated with oxidative stress and apoptosis, suggesting that MGST3 might play a role in the pathophysiology of Alzheimer's disease. Here, we report that knockdown (KD) of MGST3 in cell lines reduced the protein level of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the resulting amyloidogenesis. Interestingly, MGST3 KD did not alter intracellular reactive oxygen species level but selectively reduced the expression of apoptosis indicators which could be associated with the receptor of cysteinyl leukotrienes, the downstream metabolites of MGST3 in arachidonic acid pathway. We then showed that the effect of MGST3 on BACE1 was independent of cysteinyl leukotrienes but involved a translational mechanism. Further RNA-seq analysis identified that regulator of G-protein signaling 4 (RGS4) was a target gene of MGST3. Silencing of RGS4 inhibited BACE1 translation and prevented MGST3 KD-mediated reduction of BACE1. The potential mechanism was related to AKT activity, as the protein level of phosphorylated AKT was significantly reduced by silencing of MGST3 and RGS4, and the AKT inhibitor abolished the effect of MGST3/RGS4 on phosphorylated AKT and BACE1. Together, MGST3 regulated amyloidogenesis by controlling BACE1 protein expression, which was mediated by RGS4 and downstream AKT signaling pathway.

A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression.

Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.

A New One-Tube Reaction Assay for the Universal Determination of Sweet Cherry (Prunus avium L.) Self-(In)Compatible MGST- and S-Alleles Using Capillary Fragment Analysis.

The sweet cherry plant (Prunus avium L.) is primarily self-incompatible, with so-called S-alleles responsible for the inability of flowers to be pollinated not only by their own pollen grains but also by pollen from other cherries having the same S-alleles. This characteristic has wide-ranging impacts on commercial growing, harvesting, and breeding. However, mutations in S-alleles as well as changes in the expression of M locus-encoded glutathione-S-transferase (MGST) can lead to complete or partial self-compatibility, simplifying orchard management and reducing possible crop losses. Knowledge of S-alleles is important for growers and breeders, but current determination methods are challenging, requiring several PCR runs. Here we present a system for the identification of multiple S-alleles and MGST promoter variants in one-tube PCR, with subsequent fragment analysis on a capillary genetic analyzer. The assay was shown to unequivocally determine three MGST alleles, 14 self-incompatible S-alleles, and all three known self-compatible S-alleles (S3', S4', S5') in 55 combinations tested, and thus it is especially suitable for routine S-allele diagnostics and molecular marker-assisted breeding for self-compatible sweet cherries. In addition, we identified a previously unknown S-allele in the 'Techlovicka´ genotype (S54) and a new variant of the MGST promoter with an 8-bp deletion in the ´Kronio´ cultivar.

High expression of the ferroptosis-associated MGST1 gene in relation to poor outcome and maladjusted immune cell infiltration in uterine corpus endometrial carcinoma.

BACKGROUND: Uterine corpus endometrial carcinoma (UCEC) tightly correlates with dysregulated iron homeostasis. MGST1 (microsomal glutathione S-transferase 1) involves in the regulation of oxidative stress and plays a key role in inhibiting iron-mediated cell death in cancer cells. Hence, we aimed to illuminate the characteristics of MGST1 expression and prognosis in UCEC using bioinformatics prediction to provide novel perspectives for theoretical supplementation and ferroptosis-based immunotherapy. METHODS: We retrieved MGST1 expression data via several public data portals. The relationships between MGST1 expression and clinicopathologic characteristics as well as survival time were evaluated via multivariate methods and Kaplan-Meier survival curves. The MGST1-interacting protein-protein interaction was also established by the STRING website. The TIMER and GEPIA databases were used to illustrate the association between MGST1 expression and infiltrated immune cells. We used the MethSurv website and the UALCAN website to determine the relationship between MGST1 expression and DNA methylation. RESULTS: MGST1 overexpression in UCEC compared with normal tissues correlates with different histological types, a lack of hormone therapy and poor survival time. MGST1 interacts with several ferroptosis-related proteins. Overexpression of MGST1 was accompanied by lower levels of NK cell and CD8+ T cell infiltration, higher myeloid-derived suppressor cell infiltration and different immunocytes with corresponding markers. Hypermethylation and low promoter methylation cooperate to regulate MGST1 expression. CONCLUSION: Elevated MGST1 expression is related to tumour development and poor prognosis, as well as dysregulated infiltration of immune cells in UCEC, which can be a potential prognostic indicator and ferroptosis-based immunotherapy target.

Blockade of Platelet CysLT1R Receptor with Zafirlukast Counteracts Platelet Protumoral Action and Prevents Breast Cancer Metastasis to Bone and Lung.

Metastases are the main cause of death in cancer patients, and platelets are largely known for their contribution in cancer progression. However, targeting platelets is highly challenging given their paramount function in hemostasis. Using a high-throughput screening and platelet-induced breast tumor cell survival (PITCS) assay as endpoint, we identified the widely used anti-asthmatic drugs and cysteinyl leukotriene receptor 1 (CysLT1R) antagonists, zafirlukast and montelukast, as new specific blockers of platelet protumoral action. Here, we show that human MDA-B02 breast cancer cells produce CysLT through mechanisms involving microsomal glutathione-S-transferase 1/2/3 (MGST1/2/3) and that can modulate cancer cell-platelet interactions via platelet-CysLT1R. CysLT1R blockade with zafirlukast decreased platelet aggregation and adhesion on cancer cells and inhibited PITCS, migration, and invasion in vitro. Zafirlukast significantly reduced, by 90%, MDA-B02 cell dissemination to bone in nude mice and reduced by 88% 4T1 spontaneous lung metastasis formation without affecting primary tumor growth. Combined treatment of zafirlukast plus paclitaxel totally inhibited metastasis of 4T1 cells to the lungs. Altogether, our results reveal a novel pathway mediating the crosstalk between cancer cells and platelets and indicate that platelet CysLT1R represents a novel therapeutic target to prevent metastasis without affecting hemostasis.

MiR-379-5p targets microsomal glutathione transferase 1 (MGST1) to regulate human glioma in cell proliferation, migration and invasion and epithelial-mesenchymal transition (EMT).

BACKGROUND: Glioma is one of the most malignant tumors worldwide. This study was aimed to study the effect of miR-379-5p/MGST1 on cell proliferation, migration, invasion and EMT in glioma. METHODS: RT-qPCR detected the expression of miR-379-5p and MGST1 in RNA level in glioma cell lines. Bioinformatic analysis was made to explore the associations between miR-379-5p and MGST1 while survival analysis was made with regards to MGST1 expression in glioma patients. Western blot analysis was applied to measure the EMT changes. MTT examined the cell viability. Transwell was used to detect the cellular invasion and migration. The binding sites between miR-379-5p and MGST1 were validated by luciferase reporter assays. RESULTS: miR-379-5p expression was lower in glioma cells. MiR-379-5p increase inhibited the viability, migration, invasion and EMT while inhibition of miR-379-5p showed a reverse effect. MGST1 inhibition curbed the cell functions. MiR-379-5p targeted and regulated MGST1 expression. Lower MGST1 is related to higher survival rate. CONCLUSION: miR-379-5p could regulate glioma cell viability, migration, invasion and EMT through MGST1, suggesting that miR-379-5p/MGST1 axis might function in the regulation of glioma progression.

Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells.

Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.

Green tea epigallocatechin gallate binds to and inhibits respiratory complexes in swelling but not normal rat hepatic mitochondria.

Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5-100µM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca(2+) overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca(2+) overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.

Bioinformatics and Machine Learning Methods Identified MGST1 and QPCT as Novel Biomarkers for Severe Acute Pancreatitis.

Severe acute pancreatitis (SAP) is a life-threatening gastrointestinal emergency. The study aimed to identify biomarkers and investigate molecular mechanisms of SAP. The GSE194331 dataset from GEO database was analyzed using bioinformatics. Differentially expressed genes (DEGs) associated with SAP were identified, and a protein-protein interaction network (PPI) was constructed. Machine learning algorithms were used to determine potential biomarkers. Gene set enrichment analysis (GSEA) explored molecular mechanisms. Immune cell infiltration were analyzed, and correlation between biomarker expression and immune cell infiltration was calculated. A competing endogenous RNA network (ceRNA) was constructed, and biomarker expression levels were quantified in clinical samples using RT-PCR. 1101 DEGs were found, with two modules most relevant to SAP. Potential biomarkers in peripheral blood samples were identified as glutathione S-transferase 1 (MGST1) and glutamyl peptidyltransferase (QPCT). GSEA revealed their association with immunoglobulin regulation, with QPCT potentially linked to pancreatic cancer development. Correlation between biomarkers and immune cell infiltration was demonstrated. A ceRNA network consisting of 39 nodes and 41 edges was constructed. Elevated expression levels of MGST1 and QPCT were verified in clinical samples. In conclusion, peripheral blood MGST1 and QPCT show promise as SAP biomarkers for diagnosis, providing targets for therapeutic intervention and contributing to SAP understanding.

Coniferyl ferulate alleviate xylene-caused hematopoietic stem and progenitor cell toxicity by Mgst2.

Xylene exposure is known to induce toxicity in hematopoietic stem and progenitor cells (HSPCs), leading to bone marrow suppression and potential leukemogenesis. However, research on the gene expression profiles associated with xylene-induced toxicity in HSPCs, and effective therapeutic interventions, remains scarce. In our study, we employed single-cell RNA sequencing to capture the transcriptomic shifts within bone marrow HSPCs both prior to and following treatment with coniferyl ferulate (CF) in a mouse model of xylene-induced hematotoxicity. Subsequently, we pinpointed CF as a targeted agent using SPR-LC/MS analysis. This enabled us to confirm the link between the gene Mgst2 and specific cellular subtypes. Our data revealed that CF significantly countered the reduction of both monocyte and neutrophil progenitor cells, which are commonly affected by xylene toxicity. Through targeted analysis, we identified Mgst2 as a direct molecular target of CF. Notably, Mgst2 is preferentially expressed in neutrophil progenitor cells and is implicated in mitochondrial metabolic processes. By selectively inhibiting Mgst2 in bone marrow, we observed amelioration of xylene-induced hematotoxic effects. In summary, our findings suggest that coniferyl ferulate can mitigate the detrimental impact of xylene on hematopoietic stem and progenitor cells by targeting Mgst2, particularly within subpopulations of neutrophil progenitors. This discovery not only advances our comprehension of the cellular response of HSPCs to xenobiotic stressors like xylene but also identifies CF and Mgst2 as potential therapeutic targets for alleviating xylene-induced hematotoxicity.

Targeting MGST1 Makes Non-Small Cell Lung Cancer Cells Sensitive to Radiotherapy by Epigenetically Enhancing ALOX15-Mediated Ferroptosis.

BACKGROUND: Ferroptosis is closely related to radiotherapy resistance in multiple can-cers. Herein, the role of microsomal glutathione S-transferase 1 (MGST1) in regulating ferropto-sis and radiotherapy resistance in non-small cell lung cancer (NSCLC) was investigated. METHODS: Radiation-resistant NSCLC cells (NCI-1299-IR and HCC827-IR cells) were estab-lished. After exposure to X-ray, cell proliferation and survival were assessed by colony formation assay and CCK-8 assay, and lipid ROS level was examined by the fluorophore BODIPY™ 581/591 C11. MDA, GSH, and Fe2+ levels were measured by ELISA kits. The molecular interac-tion was analyzed using ChIP and MSP assays. RESULTS: Our results showed that RSL3 treatment greatly enhanced the radiotherapy sensitivity of NCI-1299-IR and HCC827-IR cells. It was subsequently revealed that MGST1 was highly ex-pressed in NCI-1299-IR and HCC827-IR cells than its parent cells, and silencing of MGST1 re-duced radioresistance of NCI-1299-IR and HCC827-IR cells by facilitating ferroptosis. Mechanis-tically, MGST1 knockdown greatly reduced HO-1 and DNMT1/3A protein levels, leading to re-duced DNA methylation on the ALOX15 promoter region, thereby epigenetically upregulating ALOX15 expression. As expected, the promoting effects of MGST1 silencing on radiosensitivity and ferroptosis in radiation-resistant NSCLC cells were strikingly eliminated by ALOX15 knock-down. CONCLUSION: MGST1 knockdown epigenetically enhanced radiotherapy sensitivity of NCSLC cells by promoting ALOX15-mediated ferroptosis through regulating the HO-1/DNMT1 pathway.

Morphometric Analysis of the Eye by Magnetic Resonance Imaging in MGST2-Gene-Deficient Mice.

Strabismus, a neuro-ophthalmological condition characterized by misalignment of the eyes, is a common ophthalmic disorder affecting both children and adults. In our previous study, we identified the microsomal glutathione S-transferase 2 (MGST2) gene as one of the potential candidates for comitant strabismus susceptibility in a Japanese population. The MGST2 gene belongs to the membrane-associated protein involved in the generation of pro-inflammatory mediators, and it is also found in the protection against oxidative stress by decreasing the reactivity of oxidized lipids. To look for the roles of the MGST2 gene in the development, eye alignment, and overall morphology of the eye as the possible background of strabismus, MGST2 gene knockout (KO) mice were generated by CRISPR/Cas9-mediated gene editing with guide RNAs targeting the MGST2 exon 2. The ocular morphology of the KO mice was analyzed through high-resolution images obtained by a magnetic resonance imaging (MRI) machine for small animals. The morphometric analyses showed that the height, width, and volume of the eyeballs in MGST2 KO homozygous mice were significantly greater than those of wild-type mice, indicating that the eyes of MGST2 KO homozygous mice were significantly enlarged. There were no significant differences in the axis length and axis angle. These morphological changes may potentially contribute to the development of a subgroup of strabismus.

Enzymology of reactive intermediate protection: kinetic analysis and temperature dependence of the mesophilic membrane protein catalyst MGST1.

Glutathione transferases (GSTs) are a class of phase II detoxifying enzymes catalysing the conjugation of glutathione (GSH) to endogenous and exogenous electrophilic molecules, with microsomal glutathione transferase 1 (MGST1) being one of its key members. MGST1 forms a homotrimer displaying third-of-the-sites-reactivity and up to 30-fold activation through modification of its Cys-49 residue. It has been shown that the steady-state behaviour of the enzyme at 5 °C can be accounted for by its pre-steady-state behaviour if the presence of a natively activated subpopulation (~ 10%) is assumed. Low temperature was used as the ligand-free enzyme is unstable at higher temperatures. Here, we overcame enzyme lability through stop-flow limited turnover analysis, whereby kinetic parameters at 30 °C were obtained. The acquired data are more physiologically relevant and enable confirmation of the previously established enzyme mechanism (at 5 °C), yielding parameters relevant for in vivo modelling. Interestingly, the kinetic parameter defining toxicant metabolism, kcat /KM , is strongly dependent on substrate reactivity (Hammett value 4.2), underscoring that glutathione transferases function as efficient and responsive interception catalysts. The temperature behaviour of the enzyme was also analysed. Both the KM and KD values decreased with increasing temperature, while the chemical step k3 displayed modest temperature dependence (Q10 : 1.1-1.2), mirrored in that of the nonenzymatic reaction (Q10 : 1.1-1.7). Unusually high Q10 values for GSH thiolate anion formation (k2 : 3.9), kcat (2.7-5.6) and kcat /KM (3.4-5.9) support that large structural transitions govern GSH binding and deprotonation, which limits steady-state catalysis.

Exploring the influence of magnesium stearate content and mixing modality on the rheological properties and in vitro aerosolization of dry powder inhaler.

Since carrier-based dry powder inhalers (DPIs) suffer from inadequate drug deposition in the lung, an increasing number of marketed products have added magnesium stearate (MgSt) to improve the aerosolization, dispersion, and stability against moisture of DPI. However, for carrier-based DPI, there is a lack of examination of the optimal MgSt content as well as the mixing modality, and there is also a need to verify the applicability of rheological properties to predict the in vitro aerosolization of DPI formulations containing MgSt. Therefore, in this work, DPI formulations were prepared using fluticasone propionate as a model drug and commercial crystalline lactose Respitose® SV003 as a carrier within 1% MgSt content, the effect of MgSt content on the rheological and aerodynamic properties were investigated. After the optimal MgSt content was determined, the effects of mixing modality, mixing order, and carrier size on formulation properties were further investigated. Meanwhile, correlations were established between rheological parameters and in vitro drug deposition parameters, and the contribution of rheological parameters were determined using principal component analysis (PCA). The results showed that the optimal content of MgSt in DPI formulations is 0.25%-0.5% under both high-shear and low-shear, using medium-sized carriers (D50 around 70 µm) and low-shear mixing are beneficial for improving in vitro aerosolization. Good linear relationships between powder rheological parameters such as basic flow energy (BFE), specific energy (SE), Permeability and fine particle fraction (FPF) were established, PCA showed that both flowability and adhesion are key properties affecting FPF. In conclusion, both MgSt content and mixing modality can influence rheological properties of the DPI, which can be used as a screeing tool for DPI formuluation and preparation process optimization.

Whole-Genome Sequencing Data Reveal New Loci Affecting Milk Production in German Black Pied Cattle (DSN).

German Black Pied (DSN) is considered an ancestral population of the Holstein breed. The goal of the current study was to fine-map genomic loci for milk production traits and to provide sequence variants for selection. We studied genome-wide associations for milk-production traits in 2160 DSN cows. Using 11.7 million variants from whole-genome sequencing of 304 representative DSN cattle, we identified 1980 associated variants (-log10(p) ≥ 7.1) in 13 genomic loci on 9 chromosomes. The highest significance was found for the MGST1 region affecting milk fat content (-log10(p) = 11.93, MAF = 0.23, substitution effect of the minor allele (ßMA) = -0.151%). Different from Holstein, DGAT1 was fixed (0.97) for the alanine protein variant for high milk and protein yield. A key gene affecting protein content was CSN1S1 (-log10(p) = 8.47, MAF = 049, ßMA = -0.055%) and the GNG2 region (-log10(p) = 10.48, MAF = 0.34, ßMA = 0.054%). Additionally, we suggest the importance of FGF12 for protein and fat yield, HTR3C for milk yield, TLE4 for milk and protein yield, and TNKS for milk and fat yield. Selection for favored alleles can improve milk yield and composition. With respect to maintaining the dual-purpose type of DSN, unfavored linkage to genes affecting muscularity has to be investigated carefully, before the milk-associated variants can be applied for selection in the small population.

MGST1 alleviates the oxidative stress of trophoblast cells induced by hypoxia/reoxygenation and promotes cell proliferation, migration, and invasion by activating the PI3K/AKT/mTOR pathway.

Preeclampsia (PE) is a common pregnancy-specific syndrome with an incidence of 4.6% in all pregnant women. Numerous studies have uncovered the functions and mechanisms of microsomal glutathione transferase 1 (MGST1) in different diseases and cellular processes, but whether MGST1 plays a role in PE remains unclear. Our study aimed to investigate the regulatory role of MGST1 in PE progression. In this study, the HTR8/SVneo cells were incubated with CoCl2 (250 µM) to mimic hypoxia in trophoblasts. Real-time quantitative polymerase chain reaction revealed that MGST1 was dramatically reduced in the placenta of PE patients. The proliferation of HTR8/SVneo cells was assessed via the Cell Counting Kit-8 and colony formation assays, and the results showed that MGST1 upregulation increased the cell viability of HTR8/SVneo cells. In addition, wound healing and Transwell assays unveiled that the elevation of MGST1 enhanced trophoblast cell migration and invasion. Moreover, the upregulation of MGST1 alleviated the hypoxia-induced oxidative stress in trophoblast cell. Mechanically, we found that MGST1 regulated PE progression by activating the phosphoinositide-3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway. In conclusion, MGST1 alleviated the oxidative stress of trophoblast cells induced by hypoxia/reoxygenation and promoted cell proliferation, migration, and invasion via the activation of the PI3K/AKT/mTOR pathway in PE. These results suggested that MGST1 can be a potential target for the prevention and treatment of PE.

Caveolin-1ß promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles inSpodoptera frugiperda21 insect cells.

The heterologous expression in Spodoptera frugiperda 21 (Sf21) insect cells of the ß isoform of canine caveolin-1 (caveolin-1ß), using a baculovirus-based vector, resulted in intracellular vesicles enriched in caveolin-1ß. We investigated whether these vesicles could act as membrane reservoirs, and promote the production of an active membrane protein (MP) when co-expressed with caveolin-1ß. We chose hMGST1 (human microsomal glutathione S-transferase 1) as the co-expressed MP. It belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral MPs, and, as a phase II detoxification enzyme, it catalyzes glutathione conjugation of lipophilic drugs present in the lipid membranes. In addition to its pharmaceutical interest, its GST activity can be conveniently measured. The expression of both MPs were followed by Western blots and membrane fractionation on density gradient, and their cell localization by immunolabeling and transmission electron microscopy. We showed that caveolin-1ß kept its capacity to induce intracellular vesicles in the host when co-expressed with hMGST1, and that hMGST1 is in part addressed to these vesicles. Remarkably, a fourfold increase in the amount of active hMGST1 was found in the most enriched membrane fraction, along with an increase of its specific activity by 60% when it was co-expressed with caveolin-1ß. Thus, heterologously expressed caveolin-1ß was able to induce cytoplasmic vesicles in which a co-expressed exogenous MP is diverted and sequestered, providing a favorable environment for this cargo.

Continuous direct compression: Development of an empirical predictive model and challenges regarding PAT implementation.

In this study, an empirical predictive model was developed based on the quantitative relationships between blend properties, critical quality attributes (CQA) and critical process parameters (CPP) related to blending and tableting. The blend uniformity and API concentration in the tablets were used to elucidate challenges related to the processability as well as the implementation of PAT tools. Thirty divergent ternary blends were evaluated on a continuous direct compression line (ConsiGma™ CDC-50). The trials showed a significant impact of the impeller configuration and impeller speed on the blending performance, whereas a limited impact of blend properties was observed. In contrast, blend properties played a significant role during compression, where changes in blend composition significantly altered the tablet quality. The observed correlations allowed to develop an empirical predictive model for the selection of process configurations based on the blend properties, reducing the number of trial runs needed to optimize a process and thus reducing development time and costs of new drug products. Furthermore, the trials elucidated several challenges related to blend properties that had a significant impact on PAT implementation and performance of the CDC-platform, highlighting the importance of further process development and optimization in order to solve the remaining challenges.

MGST1 is a redox-sensitive repressor of ferroptosis in pancreatic cancer cells.

Ferroptosis is a type of nonapoptotic cell death driven by lipid peroxidation. Here, we show a key role of MGST1 in inhibiting ferroptosis in cell cultures and mouse xenograft models. Ferroptosis activators induce MGST1 upregulation in human pancreatic ductal adenocarcinoma (PDAC) cell lines in an NFE2L2-dependent manner. The genetic depletion of MGST1 or NFE2L2 has a similar effect in promoting ferroptosis, whereas the re-expression of MGST1 restores the resistance of NFE2L2-knockdown cells to ferroptosis. MGST1 inhibits ferroptotic cancer cell death partly by binding to ALOX5, resulting in reduced lipid peroxidation. The expression of MGST1 is positively correlated with NFE2L2 expression in pancreatic tumors, which is implicated in the poor prognosis of patients with PDAC. These findings not only provide a valuable insight into the defense mechanism against ferroptotic cell death, but also indicate that targeting the MGST1 redox-sensitive pathway may be a promising strategy for the treatment of PDAC.

From Cyanobacteria to Human, MAPEG-Type Glutathione-S-Transferases Operate in Cell Tolerance to Heat, Cold, and Lipid Peroxidation.

The MAPEG2 sub-family of glutathione-S-transferase proteins (GST) has been poorly investigated in vivo, even in prokaryotes such as cyanobacteria the organisms that are regarded as having developed glutathione-dependent enzymes to protect themselves against the reactive oxygen species (ROS) often produced by their powerful photosynthesis. We report the first in vivo analysis of a cyanobacterial MAPEG2-like protein (Sll1147) in the model cyanobacterium Synechocystis PCC 6803. While Sll1147 is dispensable to cell growth in standard photo-autotrophic conditions, it plays an important role in the resistance to heat and cold, and to n-tertbutyl hydroperoxide (n-tBOOH) that induces lipid peroxidation. These findings suggest that Sll1147 could be involved in membrane fluidity, which is critical for photosynthesis. Attesting its sensitivity to these stresses, the Δsll1147 mutant lacking Sll1147 challenged by heat, cold, or n-tBOOH undergoes transient accumulation of peroxidized lipids and then of reduced and oxidized glutathione. These results are welcome because little is known concerning the signaling and/or protection mechanisms used by cyanobacteria to cope with heat and cold, two inevitable environmental stresses that limit their growth, and thus their production of biomass for our food chain and of biotechnologically interesting chemicals. Also interestingly, the decreased resistance to heat, cold and n-tBOOH of the Δsll1147 mutant could be rescued back to normal (wild-type) levels upon the expression of synthetic MAPEG2-encoding human genes adapted to the cyanobacterial codon usage. These synthetic hmGST2 and hmGST3 genes were also able to increase the Escherichia coli tolerance to heat and n-tBOOH. Collectively, these finding indicate that the activity of the MAPEG2 proteins have been conserved, at least in part, during evolution from (cyano)bacteria to human.