CYP74B34 Enzyme from Carrot (Daucus carota) with a Double Hydroperoxide Lyase/Epoxyalcohol Synthase Activity: Identification and Biochemical Properties.

The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.

Effects of concurrent training and N-acetylcysteine supplementation on cardiac remodeling and oxidative stress in middle-aged spontaneously hypertensive rats.

BACKGROUND: This study evaluated the effects of concurrent isolated training (T) or training combined with the antioxidant N-acetylcysteine (NAC) on cardiac remodeling and oxidative stress in spontaneously hypertensive rats (SHR). METHODS: Six-month-old male SHR were divided into sedentary (S, n = 12), concurrent training (T, n = 13), sedentary supplemented with NAC (SNAC, n = 13), and concurrent training with NAC supplementation (TNAC, n = 14) groups. T and TNAC rats were trained three times a week on a treadmill and ladder; NAC supplemented groups received 120 mg/kg/day NAC in rat chow for eight weeks. Myocardial antioxidant enzyme activity and lipid hydroperoxide concentration were assessed by spectrophotometry. Gene expression of NADPH oxidase subunits Nox2, Nox4, p22 phox, and p47 phox was evaluated by real time RT-PCR. Statistical analysis was performed using ANOVA and Bonferroni or Kruskal-Wallis and Dunn. RESULTS: Echocardiogram showed concentric remodeling in TNAC, characterized by increased relative wall thickness (S 0.40 ± 0.04; T 0.39 ± 0.03; SNAC 0.40 ± 0.04; TNAC 0.43 ± 0.04 *; * p < 0.05 vs T and SNAC) and diastolic posterior wall thickness (S 1.50 ± 0.12; T 1.52 ± 0.10; SNAC 1.56 ± 0.12; TNAC 1.62 ± 0.14 * mm; * p < 0.05 vs T), with improved contractile function (posterior wall shortening velocity: S 39.4 ± 5.01; T 36.4 ± 2.96; SNAC 39.7 ± 3.44; TNAC 41.6 ± 3.57 * mm/s; * p < 0.05 vs T). Myocardial lipid hydroperoxide concentration was lower in NAC treated groups (S 210 ± 48; T 182 ± 43; SNAC 159 ± 33 *; TNAC 110 ± 23 *# nmol/g tissue; * p < 0.05 vs S, # p < 0.05 vs T and SNAC). Nox 2 and p22 phox expression was higher and p47 phox lower in T than S [S 1.37 (0.66-1.66); T 0.78 (0.61-1.04) *; SNAC 1.07 (1.01-1.38); TNAC 1.06 (1.01-1.15) arbitrary units; * p < 0.05 vs S]. NADPH oxidase subunits did not differ between TNAC, SNAC, and S groups. CONCLUSION: N-acetylcysteine supplementation alone reduces oxidative stress in untreated spontaneously hypertensive rats. The combination of N-acetylcysteine and concurrent exercise further decreases oxidative stress. However, the lower oxidative stress does not translate into improved cardiac remodeling and function in untreated spontaneously hypertensive rats.

[Ferroptosis in the testis: Progress in research].

Ferroptosis is a form of cell death resulting from the disruption of iron metabolism within cells and excessive accumulation of lipid peroxides. Research indicates that, under the influence of various pathogenic factors, ferroptosis impacts the male reproductive system and fertility by affecting the synthesis of testicular hormones and regulation of cellular functions through different pathways and numerators. This paper provides an overview of the action mechanisms of ferroptosis in the testis and its correlation with pathogenicity, offering some new insights into the treatment of male reproductive system diseases.

"All-in-one" metal polyphenol network nanocapsules integrated microneedle patches for lipophagy fueled ferroptosis-mediated multimodal therapy.

Ferroptosis-mediated multimodal therapy has emerged as a promising strategy for tumor elimination, with lipid peroxide (LPO) playing a pivotal role. However, the therapeutic efficiency is limited due to insufficient intracellular levels of free fatty acids (FFA), which severely hinder the production of LPO. To address this limitation, we proposed a lipophagy strategy aimed at degrading lipid droplets (LDs) to release FFA, serving as the essential "fuel" for LPO production. In this study, the lipophagy inducer epigallocatechin gallate (EGCG) was self-assembled with reactive oxygen species (ROS)-producer phenethyl isothiocyanate (PEITC) mediated by Fe2+ to form EFP nanocapsules, which were further integrated into microneedle patches to form a "all-in-one" EFP@MNs. The metal-polyphenol network structure of EFP endow it with photothermal therapy capacity. Upon insertion into tumors, the released EFP nanocapsules were demonstrated to induce lipophagy through metabolic disturbance, thereby promoting LPO production and facilitating ferroptosis. When combined with photothermal therapy, this approach significantly remolded the tumor immune microenvironment by driving tumor-associated macrophages toward M1 phenotype and enhancing dendritic cell maturation. Encouragingly, in conjunction with αPD-L1 treatment, the proposed EFP@MNs exhibited remarkable efficacy in tumor ablation. Our study presents a versatile framework for utilizing microneedle patches to power ferroptosis-mediated multimodal therapy.

Thickness determination of hydroperoxidized lipid bilayers from medium-resolution cryo-TEM images.

As the primary products of lipid oxidation, lipid hydroperoxides constitute an important class of lipids generated by aerobic metabolism. However, despite several years of effort, the structure of the hydroperoxidized bilayer has not yet been observed under electron microscopy. Here we use a 200 kV Cryo-TEM to image small unilamellar vesicles (SUVs) made (i) of pure POPC or SOPC, (ii) of their pure hydroperoxidized form, and (iii) of their equimolar mixtures. We show that the challenges posed by the determination of the thickness of the hydroperoxidized bilayers under these observation conditions can be addressed by an image analysis method that we developed and describe here.

In-situ co-immobilization of lipase, lipoxygenase and L-cysteine within a metal-amino acid framework for conversion of soybean oil into higher-value products.

We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products.

Reactions of lipid hydroperoxides and how they may contribute to ferroptosis sensitivity.

The accumulation of lipid hydroperoxides (LOOHs) has long been associated with numerous pathologies and has more recently been shown to drive a specific type of cell death known as ferroptosis. In competition with their detoxification by glutathione peroxidases, LOOHs can react with both one-electron reductants and one-electron oxidants to afford radicals that initiate lipid peroxidation (LPO) chain reactions leading to more LOOH. These radicals can alternatively undergo a variety of (primarily unimolecular) reactions leading to electrophilic species that destabilize the membrane and/or react with cellular nucleophiles. While some reaction mechanisms leading to lipid-derived electrophiles have been known for some time, others have only recently been elucidated. Since LOOH (and related peroxides, LOOL) undergo these various reactions at different rates to afford distinct product distributions specific to their structures, not all LOOHs (and LOOLs) should be equivalently problematic for the cell - be it in their propensity to initiate further LPO or fragment to electrophiles, drive membrane permeabilization and eventual cell death. Herein we briefly review the fates of LOOH and discuss how they may contribute to the modulation of cell sensitivity to ferroptosis by different lipids.

The versatile CYP74 clan enzyme CYP440A19 from the European lancelet Branchiostoma lanceolatum biosynthesizes novel macrolactone, epoxydiene, and related oxylipins.

The present work reports the detection and cloning of a new CYP74 clan gene of the European lancelet (Branchiostoma lanceolatum) and the biochemical characterization of the recombinant protein CYP440A19. CYP440A19 possessed epoxyalcohol synthase (EAS) activity towards the 13-hydroperoxides of linoleic and α-linolenic acids, which were converted into oxiranylcarbinols, i.e., (11S,12R,13S)-11-hydroxy-12,13-epoxy derivatives. The conversion of 9-hydroperoxides produced distinct products. Linoleic acid 9(S)-hydroperoxide (9-HPOD) was mainly converted into 9,14-diol (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid and macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In addition, (8Z)-colneleic acid was formed. Brief incubations of the enzyme with 9-HPOD in a biphasic system of hexane-water enabled the isolation of the short-lived 9,10-epoxydiene (9S,10R,11E,13E)-9,10-epoxy-11,13-octadecadienoic acid. The structure and stereochemistry of the epoxyalcohols, macrolactone, (8Z)-colneleic acid (Me), and 9,10-epoxydiene (Me) were confirmed by 1H-NMR, 1H-1H-COSY, 1H-13C-HSQC, and 1H-13C-HMBC spectroscopy. Macrolactone and cis-9,10-epoxydiene are novel products. The 9-hydroperoxide of α-linolenic acid was mainly converted into macrolactone 9(S),10(R)-epoxy-11(E),15(Z)-octadecadiene-13(S)-olide and a minority of divinyl ethers, particularly (8Z)-colnelenic acid. The versatility of enzyme catalysis, as well as the diversity of CYP74s and other enzymes involved in oxylipin biosynthesis, demonstrates the complexity of the lipoxygenase pathway in lancelets.

Revealing the Ferroptotic Phenotype of Medulloblastoma.

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named "ferroptosis." The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic "bubbling" phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Exploring Ferroptosis-Inducing Therapies for Cancer Treatment: Challenges and Opportunities.

Conventional cancer therapies typically aim to eliminate tumor cells by inducing cell death. The emergence of resistance to these standard treatments has spurred a shift in focus toward exploring alternative cell death pathways beyond apoptosis. Ferroptosis-an iron-dependent regulated cell death triggered by lipid peroxide accumulation-has gained prominence in cancer research in recent years. Ferroptosis-inducing therapies hold promise for overcoming resistance encountered with conventional treatments. However, challenges, including the lack of distinctive ferroptosis markers and the intricate role of ferroptosis within the tumor microenvironment, currently hinder the clinical translation of these therapies. This perspective article critically outlines these hurdles and highlights unexplored opportunities in ferroptosis research, aiming to refine its therapeutic utilization in combating cancer.

Free Radical Lipid Peroxidation Induced by Reactive Halogen Species.

The review is devoted to the mechanisms of free radical lipid peroxidation (LPO) initiated by reactive halogen species (RHS) produced in mammals, including humans, by heme peroxidase enzymes, primarily myeloperoxidase (MPO). It has been shown that RHS can participate in LPO both in the initiation and branching steps of the LPO chain reactions. The initiation step of RHS-induced LPO mainly involves formation of free radicals in the reactions of RHS with nitrite and/or with amino groups of phosphatidylethanolamine or Lys. The branching step of the oxidative chain is the reaction of RHS with lipid hydroperoxides, in which peroxyl and alkoxyl radicals are formed. The role of RHS-induced LPO in the development of human inflammatory diseases (cardiovascular and neurodegenerative diseases, cancer, diabetes, rheumatoid arthritis) is discussed in detail.

Roles of Reactive Carbonyl Species (RCS) in Plant Response to Abiotic Stress.

Abiotic and biotic stress conditions lead to production of reactive carbonyl species (RCS) which are lipid peroxide derivatives and have detrimental effects on plant cells especially at high concentrations. There are several molecules that can be classified in RCS; among them, 4-hydroxy-(E)-2-nonenal (HNE) and acrolein are widely recognized and studied because of their toxicity. The toxicity mechanisms of RCS are well known in animals but their roles in plant systems especially signaling aspects in metabolism need to be addressed. This chapter focuses on the production mechanisms of RCS in plants as well as how plants scavenge and modify them to prevent irreversible damage in the cell. We aimed to get a comprehensive look at the literature to summarize the signaling roles of RCS in plant metabolism and their interaction with other signaling mechanisms such as highly recognized reactive oxygen species (ROS) signaling. Changing climate promotes more severe abiotic stress effects on plants which also decrease yield on the field. The effects of abiotic stress conditions on RCS metabolism are also gathered in this chapter including their signaling roles during abiotic stresses. Different methods of measuring RCS in plants are also presented in this chapter to draw more attention to the study of RCS metabolism in plants.

A ferroptosis amplifier based on triple-enhanced lipid peroxides accumulation strategy for effective pancreatic cancer therapy.

As an iron dependent regulatory cell death process driven by excessive lipid peroxides (LPO), ferroptosis is recognized as a powerful weapon for pancreatic cancer (PC) therapy. However, the tumor microenvironment (TME) with hypoxia and elevated glutathione (GSH) expression not only inhibits LPO production, but also induces glutathione peroxidase 4 (GPX4) mediated LPO clearance, which greatly compromise the therapeutic outcomes of ferroptosis. To address these issues, herein, a novel triple-enhanced ferroptosis amplifier (denoted as Zal@HM-PTBC) is rationally designed. After intravenous injection, the overexpressed H2O2/GSH in TME induces the collapse of Zal@HM-PTBC and triggers the production of oxygen and reactive oxygen species (ROS), which synergistically amplify the degree of lipid peroxidation (broaden sources). Concurrently, GSH consumption because of the degradation of the hollow manganese dioxide (HM) significantly weakens the activity of GPX4, resulting in a decrease in LPO clearance (reduce expenditure). Moreover, the loading and site-directed release of zalcitabine further promotes autophagy-dependent LPO accumulation (enhance effectiveness). Both in vitro and in vivo results validated that the ferroptosis amplifier demonstrated superior specificity and favorable therapeutic responses. Overall, this triple-enhanced LPO accumulation strategy demonstrates the ability to facilitate the efficacy of ferroptosis, injecting vigorous vitality into the treatment of PC.

Evaluation of Antioxidant Activity and Treatment of Eczema by Berberine Hydrochloride-Loaded Liposomes-in-Gel.

In this paper, berberine hydrochloride-loaded liposomes-in-gel were designed and developed to investigate their antioxidant properties and therapeutic effects on the eczema model of the mouse. Berberine hydrochloride-liposomes (BBH-L) as the nanoparticles were prepared by the thin-film hydration method and then dispersed BBH-L evenly in the gel matrix to prepare the berberine hydrochloride liposomes-gel (BBH-L-Gel) by the natural swelling method. Their antioxidant capacity was investigated by the free radical scavenging ability on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and H2O2 and the inhibition of lipid peroxides malondialdehyde (MDA). An eczema model was established, and the efficacy of the eczema treatment was preliminarily evaluated using ear swelling, the spleen index, and pathological sections as indicators. The results indicate that the entrapment efficiency of BBH-L prepared by the thin-film hydration method was 78.56% ± 0.7%, with a particle size of 155.4 ± 9.3 nm. For BBH-L-Gel, the viscosity and pH were 18.16 ± 6.34 m Pas and 7.32 ± 0.08, respectively. The cumulative release in the unit area of the in vitro transdermal study was 85.01 ± 4.53 µg/cm2. BBH-L-Gel had a good scavenging capacity on DPPH and H2O2, and it could effectively inhibit the production of hepatic lipid peroxides MDA in the concentration range of 0.4-2.0 mg/mL. The topical application of BBH-L-Gel could effectively alleviate eczema symptoms and reduce oxidative stress injury in mice. This study demonstrates that BBH-L-Gel has good skin permeability, excellent sustained release, and antioxidant capabilities. They can effectively alleviate the itching, inflammation, and allergic symptoms caused by eczema, providing a new strategy for clinical applications in eczema treatment.

Emerging mechanisms of ferroptosis and its implications in lung cancer.

ABSTRACT: Lung cancer is one of the most common malignancies and has the highest number of deaths among all cancers. Despite continuous advances in medical strategies, the overall survival of lung cancer patients is still low, probably due to disease progression or drug resistance. Ferroptosis is an iron-dependent form of regulated cell death triggered by the lethal accumulation of lipid peroxides, and its dysregulation is implicated in cancer development. Preclinical evidence has shown that targeting the ferroptosis pathway could be a potential strategy for improving lung cancer treatment outcomes. In this review, we summarize the underlying mechanisms and regulatory networks of ferroptosis in lung cancer and highlight ferroptosis-targeting preclinical attempts to provide new insights for lung cancer treatment.

Biodegradable zwitterionic polymer-cloaked defective metal-organic frameworks for ferroptosis-inducing cancer therapy.

Ferroptosis inhibits tumor growth by iron-dependently accumulating lipid peroxides (LPO) to a lethal extent, which can result from iron overload and glutathione peroxidase 4 (GPX4) inactivation. In this study, we developed biodegradable zwitterionic polymer-cloaked atorvastatin (ATV)-loaded ferric metal-organic frameworks (Fe-MOFs) for cancer treatment. Fe-MOFs served as nanoplatforms to co-deliver ferrous ions and ATV to cancer cells; the zwitterionic polymer membrane extended the circulation time of the nanoparticles and increased their accumulation at tumor sites. In cancer cells, the structure of the Fe-MOFs collapsed in the presence of glutathione (GSH), leading to the depletion of GSH and the release of ATV and Fe2+. The released ATV decreased mevalonate biosynthesis and GSH, resulting in GPX4 attenuation. A large number of reactive oxygen species were generated by the Fe2+-triggered Fenton reaction. This synergistic effect ultimately contributed to a lethal accumulation of LPO, causing cancer cell death. The findings both in vitro and in vivo suggested that this ferroptosis-inducing nanoplatform exhibited enhanced anticancer efficacy and preferable biocompatibility, which could provide a feasible strategy for anticancer therapy.

Role of ferroptosis and ferroptosis-related long non'coding RNA in breast cancer.

Ferroptosis, a therapeutic strategy for tumours, is a regulated cell death characterised by the increased accumulation of iron-dependent lipid peroxides (LPO). Tumour-associated long non-coding RNAs (lncRNAs), when combined with traditional anti-cancer medicines or radiotherapy, can improve efficacy and decrease mortality in cancer. Investigating the role of ferroptosis-related lncRNAs may help strategise new therapeutic options for breast cancer (BC). Herein, we briefly discuss the genes and pathways of ferroptosis involved in iron and reactive oxygen species (ROS) metabolism, including the XC-/GSH/GPX4 system, ACSL4/LPCAT3/15-LOX and FSP1/CoQ10/NAD(P)H pathways, and investigate the correlation between ferroptosis and LncRNA in BC to determine possible biomarkers related to ferroptosis.

Ferroptosis as a potential therapeutic target for age-related macular degeneration.

Cell death plays a crucial part in the process of age-related macular degeneration (AMD), but its mechanisms remain elusive. Accumulating evidence suggests that ferroptosis, a novel form of regulatory cell death characterized by iron-dependent accumulation of lipid hydroperoxides, has a crucial role in the pathogenesis of AMD. Numerous studies have suggested that ferroptosis participates in the degradation of retinal cells and accelerates the progression of AMD. Furthermore, inhibitors of ferroptosis exhibit notable protective effects in AMD, underscoring the significance of ferroptosis as a pivotal mechanism in the death of retinal cells during the process of AMD. This review aims to summarize the molecular mechanisms of ferroptosis in AMD, enumerate potential inhibitors and discuss the challenges and future opportunities associated with targeting ferroptosis as a therapeutic strategy, providing important information references and insights for the prevention and treatment of AMD.

Icariin, astragaloside a and puerarin mixture attenuates cognitive impairment in APP/PS1 mice via inhibition of ferroptosis-lipid peroxidation.

Alzheimer's disease (AD) is a neurodegenerative disease that seriously threatens the quality of life of the elderly. Its pathogenesis has not yet been fully elucidated. Ferroptosis, a cell death caused by excessive accumulation of iron-dependent lipid peroxides, has been implicated in the pathogenesis of AD. Uncontrolled lipid peroxidation is the core process of ferroptosis, and inhibiting lipid peroxidation of ferroptosis may be an important therapeutic target for AD. Based on previous studies, we mixed standards of icariin, astragaloside IV, and puerarin, named the standard mixture YHG, and investigated the effect of YHG on ferroptosis -lipid peroxidation in APP/PS1 mice. DFX, a ferroptosis inhibitor, was used as a control drug. In this study, APP/PS1 mice were used as an AD animal model, and behavioral experiments, iron level detection, Transmission electron microscopy (TEM) observation, lipid peroxidation level detection, antioxidant capacity detection, immunofluorescence, Western blot and real-time qPCR were performed. It was found that YHG could reduce body weight, significantly improve abnormal behaviors and the ultrastructure of hippocampal neurons in APP/PS1 mice. The results of biochemical tests showed that YHG reduced the contents of iron, malondialdehyde (MDA) and lipid peroxide (LPO) in brain tissue and serum, and increased the levels of superoxide dismutase (SOD) and reduced glutathione (GSH). Immunofluorescence, WesternBlot and real-time qPCR results showed that YHG could promote the expression of solute carrier family 7 member 11 (SLC7A11), solute carrier family 3 member 2 (SLC3A2) and glutathione peroxidase 4(GPX4). Inhibited the expression of long-chain acyllipid coenzyme a synthetase 4(ACSL4) and lysophosphatidyltransferase 3 (LPCAT3). This study suggests that the mechanism by which YHG improves cognitive dysfunction in APP/PS1 mice may be related to the inhibition of ferroptosis-lipid peroxidation.

A tangible method to assess native ferroptosis suppressor activity.

Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms.